BIO 362 Exam 1 Package

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BIO 362
Exam 1 Study Guide
SBU
Spring 2014
Marcu
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BIO 362 Exam 1 Review of Lecture Transcribed and Learning

Objectives Answered:
LECTURE 1: Amino Acid Biosynthesis Learning
Objectives:
Fixation of atmospheric nitrogen into biologically usable forms
N2 to NH4 to Amino Acids to Nucleotides!!!! Synthesis of
Non-Essential Amino Acids
Carbon donors: Reactions involving addition of 1 carbon.
Amino Acids are precursors of and are themselves bioactive molecules.
Why are Amino Acids important?
Building blocks of proteins (polymers of amino acids)
Precursors of other essential molecules
Minor modifications of amino acids produce potent biologically active
molecules!
Amino Acids themselves are BIOACTIVE (have biological activity)
Question: How are elements in the atmosphere incorporated into
biologically useful molecules?
So the composition of the atmosphere is mostly nitrogen. Nitrogen is
incorporated into Biologically useful molecules by nitrogen fixation.
(Diazotrophs: Are bacteria that fix Nitrogen into Biologically useful
molecules with the Enzyme Nitrogenase) .
Side Note: Photosynthesis and Sugar Biosynthesis Carbon dioxide and water
are incorporated into Sugar (Glucose ).
The Nitrogen Cycle: You take N2 and 8 H+ and get two Ammonia this
happens 8 times where it requires 16 ATPs.
Amino vs. a-Keto Acids: The only difference between Amino Acids and
AKETO ACIDS is the presence of an amine group in the amino acid.
Summary Slide:
N2 goes to NH3(ammonia) through enzyme Nitrogenase. Ammonia gets
incorporated into a-ketoglutarate to form Glutamate, the NH3 from
glutamate get be donated to other a-keto acids to form other Amino acids.
If Glutamate donates Amine group it will be converted to a-ketoglutrate. So
again ammonia made by bacteria is secreted into the soil picked up by plants
so then how is this Ammonia attached to a-ketoglutarate to form gluatamateNH3+.
Slide: How Does Ammonia become incorporated into Amino Acids?

Cycle that occurs in plants & microbes (does not occur in animals)
Plants and Microbes have Glutamine Synthetase = GS and GOGAT =
Glutamate Synthase.
EMPHASIZE that GLUTAMINE SYNTHETASE(GS) is found in
Mammals and Humans!
GOGAT IS NOT(GLUMATE SYNTHETASE)
Ammonia is coupled to a-ketoglurate to form glutamate.
First Enzyme (Glutamine Synthetase )GS will take pre-existing glutamate
and fused ammonia onto the side chain to form Glutamine. Ammonia adds to
side chain of Glutamate to form Glutamine. Glutamine is now a substrate for
the enzyme GOGAT (GLUTAMATE SYNTHETASE) . You take
ammonia from glutamine and add it to a-ketoglurate to form glutamate.
Glutamine is reconverted back to Glutamate. Glutamate is converted to
Glutamine, and then back to Glutamate (NO NET BIOSYNTHESIS OF
GLUTAMINE AND GLUTAMATE) And Remember that GOGAT is not
present in mammals.
So you are shuttling ammonia (NH3) adding it to Glutamate then converted
it Glutamine then transferred to a-ketoglutarate and then to Glutamate again.
Glutamate and Glutamine they cycle to couple ammonia to a-ketoglurate to
from glutamate.
Once you form Glutamate, you can donate amine group to other a-keto-acids
to form corresponding amino acids.
Glutamate can be used as a substrate by enzymes called (Transaminases)
(HOW CAN THIS BE USED CLINICALLY TO MONITER LIVER
FUCNTION)
Transaminases: Will take Amine group from Glutamate and will transfer it
to an a-keto-acid. Once you strip away amine group from Glutamate it is
converted back to a-ketoglurate (a-keto acid will then be converted to
corresponding Amino Acid.) These Transaminases use Vitamin B6 as a
cofactor! In the Transaminase mechanism the main point is that it is
BiDirectional)!
Concept of Non-Essentials Amino Acids vs. Essential Amino Acid NonEssential: Humans can synthesize from pre-existing molecules, through
simple Transamination reaction, we are able to synthesize Amino Acids.
Essential Amino Acids: We need to obtain from diet.
How Transaminase Reaction is used to synthesize NON-ESSENTAIL
AMINO ACIDS? So Glutamate serve as an amine group donor to an a-keto
acid, in the process Glutamate is converted to a-ketoglurate.
Example: Alanine AminoTransferase will use Glutamate as a substrate and
transfer amine group and add it to pyruvate in the process pyruvate become
alanine.
Example: Also can use Oxaloacetate, use Glutamate as donor and convert
Oxaloacetate to Aspartate.
Also Glutamate is a substrate for Glutamine Synthetase which will take free
ammonia and conjugate it to side chain of Glutamate to form Glutamine.
Also Glutamine itself can be used as an Amine group donor, in
ASPARAGINE SYNTHETASE which will abstract amine group from
Glutamine and add it to Aspartate in which Glutamine will get converted
back to Glutamate and you form Asparagine.
Another example of a Transamination reaction is the synthesis of Proline
and Arginine FROM GLUTAMATE.
When you see GLUTAMATE GOING TO a-KETOGLUTERATE think
of a Transamination reaction.
Important concept of Carbon donors: They donate one carbon whether its
in the form of Methyl Group, Methylene Group, or another molecule.
Cell possess several carbon donors: THF, SAM, BIOTIN.
THF can donate methylene group between Ns carbon.
S-Adenosyl Methionine can donate Methyl group to another molecule.
Why is tetra-hydrofolate (THF) Important?
THF is used to synthesize amino acids, nucleotides, cell division synthesize
through folate (Vitamin B9). Folate is a substrate for enzyme dihydrofolate
Reductase( Reduction Reaction) will take folate and convert it to
Tetrahydrofolate(THF).
THF has to be given a methyl group (charged). In one reaction Serine gives
methyl group to (N5, N10 Methylene THF). Folic Acid deficiency associated
with BIRTH DEFECTS such as anencephaly and spina bifida.
Emphasized! MTHFR Reaction.

N5, N10 Methylene THF TO N5 Methyl THF (By Methylene
tetraHydrofolate Reductase) (MTHFR)
(MTHFR): Many polymorphisms in this enzyme, polymorphisms in
MTHFR reduce its activity. Most common mutation in 677Cytosine to
thymine. Pregnant women should check if they have a mutation in MTHFR.
Polymorphisms in (MTHFR) are linked to fetal loss, anencephaly, spina
bifida and other disorders.
Remember Methionine can be converted to SAM in the presence of ATP.
Methionine is essential Amino acids! SAM (S-Adenosylmethionine) can
donate its methyl group to other molecules and in the process the SAM is
converted to (S-Adenysl-HomoCysteine) then can be converted to
HomoCysteine, then the molecule N5 Methyl THF comes into play.
(N5Methyl THF) is critical to convert Homocysteine to Methionine, methyl
group that THF has can be donated to Homocysteine to generate
Methionine.
Methionine is essential amino acids, only way to metabolize methionine is
via its conversion to SAM.
Another example why SAM is important is that DNA can be methylated.
Cytosine can be methylated by an enzyme called (DNA Methlytransferases)
ALL DNA METHYLATION REACTIONS REQUIRE SAM!!!!!! See why
SAM is important.
From Slide: Methylation of Cytosine in CpG repeats by DNA
methyltransferases(DNMT) reduce Gene Transcription.
But THF is important to regenerate SAM, if you dont have N5-Methyl THF
you cant regenerate SAM and you will have all sorts of problems in
Methylation reactions!
Another example where SAM is important (Histamine in inflammatory
response (Enzyme Histamine N-MethlyTransferase will transfer methyl
group onto Histamine, take methyl group from SAM, add Methyl group to
Histamine and convert it to an inactive form.
Homocysteine can be further metabolized and accumulate.
YOU MUST KNOW THAT HOMOCYSTEINE IS A PRECURSOR OF
CYSTEINE!
Question: Why do MTHFR Mutations lead to elevated levels of
Homocysteine?
MTHFR MUTATIONS lead to elevated levels of HOMOCYSTEINE

because (N-Methyl THF) is important to convert Homocysteine to
Methionine.
What is important of Homocysteine is that it is a marker for cardiovascular
disease.
Homocysteine is converted to Cysteine! Individuals harboring mutations in
MTHFR posses elevated levels of Homocysteine, which is converted, to
cysteine. In platelets, cysteine metabolism generates hydrogen sulfide that
induces platelet aggregation! (Thrombosis)
Certain Amino Acids we cant synthesize (Essentials Amino Acids) we cant
synthesize them because we lack an enzyme.
An example is the biosynthesis of Lysine, Threonine, Methionine! The
Enzyme aspartokinase(not found in mammals) converts Aspartate to Lysine,
Threonine, Methionine. (Humans lack this enzyme.) Other Products of
Amino Acid Metabolism:
Heme, Bioactive Amines, Bioactive N-acyl Amino Acids
Heme BioSynthesis: Begins with Glycine, Glycine used as a precursor for
Heme.
Heme Degradation: You can get a molecule called Billirubin! This is
excreted to the large intestine and converted to Urobilinogen. In the large
intestine, Urobilinogen can be converted to Stercobillin(pigment which is
brown) Urobilinogen can also be converted in the kidney to Urobilin which
is yellow.
Jaundice: Caused by excess of Bilirubin (Heme Degradation product)
Liver dysfunction and bile duct obstruction) Jaundice can be treated by
exposing the kid to fluorescent light that isomerizes Billrubin into isomers
that can be excreted and degraded.
Bioactive Amines in which Biosynthesis involves decarboxylation of Amino
acids. (THIS IS THE ONLY IMPORTANT PART)
Precursor of Dopamine is L-Dopa, once you decarboxylate L-dopa you
convert it to Dopamine. Dopamine can be a substrate and then produce
Norepinephrine.
SAM is needed to convert Norepinephrine to Epinephrine. (Again will be a
methylation reaction)
Parkinsons Disease is characterizing by deficiency in Dopamine. This is
treated with supplementation of L-DOPA immediate precursor of
DOPAMINE.
GABA and Histamine:

Glutamate precursor of GABA (major neurotransmitter in the Central
Nervous System.)
Histamine can be synthesized from Histidine (pretty Inert) which involves
decarboxylation reaction.
Histindine Decarboxylase deficiency can cause Tourette syndrome. (Think
Deuce Bigilo male gigalo) Histamine is also an important Neurotransmitter
in the brain.
Amino Acids are Bioactive Molecules
Glutamate, Glycine and Aspartate. All three amino acids function as
neurotransmitters in the central nervous system.
Know that Amino Acids themselves can be conjugated to Lipids. Ex.
N-oleyl Serine (Regulation of Bone mass), N-Arachidonoyl Glycine
(Modulates glycine signaling in CNS).
Lecture 2: Amino Acid Degradation

Main point want to get across is that Ammonia is toxic!
Protein Degradation: How polymers of Amino Acids are broken down
into Amino Acids
Digestive Enzymes in the Stomach
Endocytosis and lysosomal degradation
Autophagy
Ubiquitin-Proteasome System.
Amino Acid Degradation
Urea Cycle How Urea Cycle is important in medicine and how enzymes we
discuss are part of the Urea Cycle and used clinically.
Learning Objectives:
Know the pathways for cellular protein degradation
Ex.) Autophagy, Ubiquitin-Proteasome pathway, digestive enzymes,
endocytosis.
Understand how transaminases contribute to amino acid degradation
Role of Alanine and Glutamine in Blood Nitrogen Transport
Understand how the Urea cycle contributes to nitrogen elimination
Review of Lecture 1:
How Nitrogen is incorporated into Amino Acids?
N2 converted to ammonia by bacteria, then ammonia taken up by the soil by
bacteria and plants then incorporated into Glutamate. Glutamate can be a
source for nitrogen and can be used to synthesize other amino acids through
transamination reactions. Transamination reactions occur both in plants and
mammals.
Synthesis of Non-Essential Amino Acids: Important to know!
Glutamine itself can be used as a source of Nitrogen, such as in Asparagine

Synthetase.(with molecule of ATP) We discuss Carbon Donors, THF AND
SAM.
THF IS charge with a Carbon from serine and you form N5,N10Methylene
THF and sub sequentially with the enzyme MTHFR you convert (N5,N10
Methylene THF to N5 Methyl THF.
Why is N5 Methyl THF important again? N5 Methyl THF is important again
because of the SAM cycle. N5 Methyl THF used to convert Homocystiene to
Methionine.
For Lecture 2 the basic theme is that ammonia is toxic!
Why is Glutamate is going to be important in Amino Acid Degradation?
Ammonia is not going to be secreted by tissues in the bloodstream. Tissues
will use Amino Acid carriers of ammonia, specifically Alanine and
Glutamine. We need to understand how the Urea Cycle contributes to
Nitrogen elimination.
Macromolecular protein degradation, proteins can be taken up by cells by
process of endocytosis will then form food vacuole or endosome then broken
up by Lysosomes, Lysosomes has a number of digestive enzymes in which
proteins can be degraded into component parts. Cells have other mechanisms
of breaking down proteins, a very important process called Autophagy (Cells
have the ability to eat their own proteins when energy is scarce, there are
certain growth factors that signal to the cell that food is plentiful and cell
should grow this signaling cascade activates a protein called mTOR which
constitutively blocks autophagy! When food is scarce, growth factors
become scarce, mTOR is no longer active as it should be and the cell
undergoes a default pathway and that is to undergo Autophagy (which could
be a number of things such as breaking down cellular components
(organelles)
Rapamycin: Rapamycin is a chemical inhibitor of mTOR and induces
autophagy. Used clinically as an immunosuppressant to prevent organ
rejection following kidney transplant it may be beneficial for people for
suffering from Alzheimers disease.
Large subset of cellular proteins are broken down through

(UbiquitinProteasome System.) the cell has to have a mechanism in which it
tells the Proteasome this protein has been tagged for breakdown. You need a
Control System, the control system is this protein called Ubiquitin, Ubiquitin
is going to be conjugated to the protein you need to breakdown, conjugated
to lysines on target proteins via its C-TERMINUS of Ubiquitin targeted to
Lysine on target proteins.
Conjugation of Ubiquitin to a Target Protein:
Ubiquitin is first conjugated to a protein called E1, in the presence of ATP;
Ubiquitin is going to be conjugated to Cysteine of E1 protein. E1 protein
will now be Ubiquitinated (not target protein) a protein in which Ubiquitin is
first attached.(Not target protein) Then protein E2-SH will pick up Ubiquitin
from E1 and Ubiquitin will be transfer to E2 then you get Ubiquitanted E2
protein. The E2 protein in the presence of Ubiquitin forms a dimer with a
protein called E3, E3 protein confers specificity and target selectively. In the
presence of E3, this complex can then recognize a substrate protein and
tagged it with Ubiquitin. Recognizing the substrate with E3 Ubiquitin is then
transfer to the substrate. This process is repeated a number of times, several
cycles until protein is poly-ubiquitinated. Protein that is poly-ubiquitenated
will be tagged for degradation. Ubiquitin is interesting because depending on
branching of Ubiquitin, protein will have different faiths. For purpose of
BIO 362 the protein that is polyubiquitinated will then be tagged for
degradation.
Conjugation of Ubiquitin to a Target Protein
Massive Complex called the Proteasome(Has multiple proteins associated

with it.) Essentially a garbage collector. 20S proteasome is the core (Engine)
two caps 19S Caps, which in essence allow cargo-targeted protein to be feed
into the proteasome. The 19S cap governs entrance of substrates.
Proteosome has open and close conformations.
The Proteasome (20S) contains three active sites localized to Beta-subunits,
Threonine Residues mediate peptide bond Hydrolysis.
19S Caps of Proteasome have lids, when a substrate comes in that is
polyubiquitnated the 19S cap will recognize it, it will open up, will have the
protein unfolded and fed into the proteasome. The 19S cap recognizes
Ubiquitinated proteins unfolds them and feed them into the protease complex
of the proteasome for Hydrolysis.
Amino Acids are broken down to ammonia and Carbon skeletons. Carbon
skeletons can be recycled, it can be pyruvate, ketone bodies(also used for
energy etc. Ammonia is being converted to UREA.
However the Nitrogen must be excreted.
Glutamine and Alanine are ammonia carriers in the blood.
Fate of Free Amino Acids: Following degradation of dietary or endogenous
proteins, the resulting amino acids can be incorporated into new proteins or
can be converted to Urea and excreted.
Composition of Urine: 80% of excreted Nitrogen is in the form of Urea!
The Nitrogen Species in the Urine:
Urea, Uric Acid and Ammonia
Urea is predominant Nitrogenous species in Urine. Urea is the breakdown

product of Amino Acids. Uric Acid is the breakdown product of Nucleotides.
The main point is that most Nitrogen is excreted in the form of UREA!
Plasma concentration of ammonia is really low, again because ammonia is
toxic. There is only really low concentration of free ammonia in the Urine.
(IT IS CRITICAL TO UNDERSTAND THAT THE UREA CYCLE ONLY
OCCURS IN THE LIVER!) Urea Cycle only occurs in the Liver. Amino
acids broken down into Urea in the liver and the Urea will be transported in
the blood and filtered out by the kidneys.
Urea will be transported in the blood and will be filtered out by the kidneys
and then excreted out. SO Urea is produced in the liver, filtered out by the
kidneys and then goes to Urine.
Logic of Amino Acid Degradation: Glutamate is source of Nitrogen for a
number of Transamination reactions. Glutamate is up the pyramid and it
donates it Nitrogen to other Amino Acids. In the case of Amino Acid
Breakdown, it will be the reverse GLUTAMATE will be the ultimate
ACCEPTOR! Take other amino acids and shuttle them to Glutamate! Single
point of Nitrogen into the Urea Cycle that will be Glutamate. Take amino
acids, metabolites and funnel them to production of Glutamate. From that
Glutamate, Nitrogen can be removed and converted to Urea.
Logic of Amino Acid Degradation:
Amino Acid Deamination: Deamination reactions are carried out by

Transaminases a-ketoglutarate is the primary amino group acceptor. A
number of Amino Acids will donate their amine group to a-ketoglutarte to
form Glutamate. Funneling all the Nitrogen to production of Glutamate in
the liver. In the liver there will be an enzyme that will be able to cleave the
amine group from Glutamate and liberate free ammonia.
An enzyme called Glutamate Dehydrogenase, which is very important
enzyme once you funnel Nitrogens into Glutamate that is a substrate for
Glutamate Dehydrogenase, which in essence cleaves bond, liberates free
ammonia and liberates a-ketoglutarate!
So you have taken all the Nitrogen from amino acids, converted them and
transferred them into Glutamate, Glutamate has a single enzyme that will
cleave ammonia out Glutamate to generate a-ketoglurate and free
ammonia! (Logic Makes sense.) Very dedicated Glutamate Dehydrogenase!
Glutamate Dehydrogenase will generate free ammonia and a-ketoglurate.
Free Ammonia will enter Urea Cycle in the liver (Again only in the
Liver)
GLUTAMATE DEHYDROGENASE:
Expressed in liver and kidneys (and other organs as well) Oxidatively
deaminates glutamate
Eliminates amino groups donated to glutamate from transamination
reactions.
Urea Cycle: Mechanism of excreting excess Nitrogen from the Body, Urea
is the ultimate carrier of Nitrogen.
Must know the source of Nitrogen in Urea! The Carbon will come from
Bicarbonate; Nitrogen in Urea is coming from (Free Ammonia and
Aspartate) the free ammonia is coming from Glutamate from the
Glutamate Dehydrogenase Step!
Urea Cycle:
Mechanism for excreting excess nitrogen from the body; Urea is the ultimate
carrier of nitrogen.
First Step we discussed is this transamination reaction, where you have
aketoglutarate and amine groups from other amino acids to form Glutamate,
those amino acids will become a-keto acids, then Glutamate will be a
substrate for Glutamate Dehydrogenase, cleaves bond from Glutamate to
generate free ammonia and regenerate a-ketoglurate. a-ketoglutarate to

Glutamate is a cycle, bringing in amine groups from other amino acids ,
funneling them to Glutamate , Glutamate dehydrogenase steps occurs free
ammonia is liberated , The free ammonia that is generated here will enter
mitochondria and will be a substrate for an enzyme called (Carbamoyl
Phosphate Synthetase(must know)!)
This combines free ammonia with BiCarbonate into a compound called
Carbamoyl Phosphate! Once this is formed the Carbamoyl phosphate will be
a substrate for the enzyme Ornithine Transcarbamoylase . This will take a
pre-existing molecule of Ornithine and couple it to Carbamoyl phosphate
and will generate a molecule called Citrulline. Citrulline will be exported
out of Mitochondria into cytoplasm.
This Citrulline is going to be a substrate for Arginosuccinate Synthetase,
which conjugates a molecule of Citrulline to a molecule of Aspartate. What
this does is that it forms an Arginiosuccinate molecule. Argniosuccinate will
be a substrate for Arginosuccinase in which its important to know that you
generate Arginine.
The last step in the UREA CYCLE is there is this enzyme called Arginase it
cleaves bond between carbon and nitrogen and generates Urea and carbon
skeleton is regenerated as Ornithine, Ornithine goes back to Mitochondria
and the cycle is repeated again.
In essence the Urea cycle ammonia is funneled through a number of
reactions to form Urea!
Kinetics of Urea
Cycle Production:
How quick the Urea Cycle goes, if you were to ingest a molecule of
ammonia, within a few seconds that ammonia will dissipate in the blood, and
within ten seconds most of it will be converted to Urea. Within ten seconds
bowls of ammonia will be converted to Urea (Very Rapid) the reason it is
rapid is because ammonia is quite TOXIC.
Urea Cycle Disorders: The two most common mutations of people who
have inborn genetic disorders of Urea. Mostly Carbomoyl phosphate
Synthetase and Ornithine Transcaramoylase, people who have defects in
these enzymes are unable to produce Urea! Arginase can also be in inborn
genetic disorder!
FOCUS ON FIRST TWO ENZYMES
1. Carbamoyl phosphate synthetase
2. Ornithine TransCarbamoylase
People who have deficits in these enzymes are unable to produce Urea! What
would happen if person were missing Caramoyl phosphate Synthetase, What
would happen to certain metabolites in these people?
Glutamate will go up, free ammonia will go up. If you cant convert
Ammonia into Urea its level will rise. Also Glutamate and ammonia are
substrates for another enzyme that enzyme is Glutamine Synthetase. So
you will also see a rise in the levels of Glutamine!
TWO TELL TALE SIGNS THAT PEOPLE ARE BORN WITH INBORN
DISORDERS OF UREA CYCLE:
1.Elevation in AMMONIA LEVELS.
2.Elevation in GLUTAMINE LEVELS.
Urea Cycle sort of like a FUNNEL.
Why is Ammonia toxic?
Ammonia converted to Glutamine (Classic Explanation). Buildup of
Glutamine results in Astrocyte swelling and compromises astrocyte function,
we dont really know correct answer we just know its toxic.
Alternative theory is that ammonia will inhibit potassium transporter in
astrocyte, potassium transporter will cause chloride ions to enter neurons.
Cl- which is present in high levels outside the cell, typically enter neuron
(inhibits neural signaling, when you have buildup of Cl- ions inside the
neuron, you flip the potential and these neurons become excitatory develop
seizures!
Ammonia triggers neural disinhibit ion and seizures by impairing astrocyte
potassium buffering.
Urea Cycle disorders: Treatments

People, who have inborn-defects of Urea cycle, can be treated successfully.
One treatment is too use PhenylButyrate .
So when there is a defect in the Urea Cycle, the glutamate will be converted
to glutamine, people given phenylbutyrate can handle glutamine it
converted to a product they can excrete. Another way to treat Urea Cycle
disorders is to keep their Amino Acid intake as low as possible!!!!! People
can also be given Benzoate, ammonia can also converted to Glycine and
then excreted through other pathways. Urea Cycle disorders is not fatal if
treated properly!
So how is ammonia transported out peripheral tissues, such as muscle, for
example?
How is ammonia generated in the Muscle to be transported back out to the
liver for excretion and production into Urea?
Because we cant just release Ammonia, cells have evolved a way to use
amino acids to transport ammonia in the blood, vehicles for ammonia in the
blood. Becomes a safe way to transfer ammonia from tissues to liver and too
kidneys as well!
Glutamine and Alanine are Nitrogen Carriers!
Glucose-Alanine cycle is a way by which our tissues transport Nitrogen to
the liver! ALT: Alanine Transaminase : Takes amines groups from glutamate
through transamination reaction and transfers them to pyruvate so you are
producing Alanine in the muscle tissues. In Muscle tissue you are funneling
nitrogen into alanine, then Alanine is secreted into the bloodstream and is
picked up by the liver, enters the liver, then same enzyme Alanine
Transminase in liver converts alanine back into Pyruvate (through reverse
reaction). Amine group in Alanine is transferred back into aketoglurate to
reform glutamate. Glutamate that is form in the liver then is funneled into
the Urea Cycle. Alanine used a carrier for Nitrogen in the blood, carrying
Nitrogen from the muscle into the liver, where its is converted back to
pyruvate, through gluconeogenesis becomes glucose than can go back to
muscle! This way transporting Alanine into the Liver and excreting Nitrogen
from the muscle to the liver.
GlucoseAlanine Cycle:
In addition:
Glutamine is also used as a nitrogen carrier. Majority of Glutamine goes to
the kidney. In the Kidney there are two enzymes responsible for breaking
down Glutamine, first one is called Glutaminase (Breaks down Glutamine,
liberates free ammonia and regenerates Glutamate!). Glutamate is then a
substrate for the same enzyme we discussed in the Liver. Glutamate
Dehydrogenase that converts glutamate to a-ketoglutarate and generates
another molecule of free ammonia.
Urobillin in Urine, if you are dehydrated,Urine becomes more concentrated.
(Urobullin will become much more pronounced.
In the kidney, Glutamine is transported out of muscle and is going to cleave
by two enzymes Glutaminase followed by Glutamate Dehydrogenase and
this will liberate free ammonia.
IMPORTANT: The free Ammonia the majority is from the KIDNEY from
production of ammonia in the Kidney through Glutamine Hydrolysis.
Remember Kidney CANNOT convert Ammonia into Urea! That ammonia
produced in the Kidney is directly secreted out.
In the Liver you generate Urea, which is absorbed by the kidney and secreted
out. Metabolism of Glutamine to Glutamate is a source for a number of
intermediates in the Krebs Cycle. A lot of Cancer Cells depend on Glutamine
for survival.
Transaminases as markers for tissue damage
AST: Aspartate Transaminase
ALT: Alanine Transaminase
When tissues are damage they release Transaminases into circulation.
Elevated levels of Transaminase are markers for tissue damage, (especially
Liver damage generally) Acetaminophen is Tynnol, which can be toxic.
Liver is damage and it starts releasing Transaminases. Treatment of animals
with Acetaminophan caused an up regulation of ALT and AST levels. Tissue
is damage and then Liver starts releasing transaminases.
Failed clinical drug trial: Telcagepant
Calcitonin gene related peptide (CGRP) is a potent vasodilator migraines are
caused by vasodilation. Telecagepant blocks the effects of CGRP
But Telcagepant elevated levels of hepatic alanine transaminase in 1.7% of
participants. Clinical trail was discontinued. Shows you the importance of
this test in which elevated levels of AST and ALT levels show tissue
damage.
In the liver Glutamate dehydrogenase produces free ammonia that is
subsequently fed into the Urea Cycle. Consequently, absence of this
enzyme would significantly reduce urea production. IN CONTRAST
Glutaminase does not significantly contribute to urea production in the liver
but it important for glutamine hydrolysis in the kidney.
Urea can be measured as well (BUN) Blood Urea Nitrogen. Which is a direct
measure of Urea in the blood. Urea produced in the Liver, secreted out into
the blood stream, can measure how much Urea is in the blood itself.
Cisplatin (Chemotherapy Agent) Found elevated levels of Blood Urea
Nitrogen the conclusion here is that there are Renal Pathologies
(Dysfunction of Kidneys)
Why would Blood Urea Nitrogen (BUN) ELEVATION be a marker for renal
dysfunction? Means kidneys arent properly filtering the blood; they arent
properly filtering out Urea out of the blood. THUS you see an elevation of
Blood Urea Nitrogen.
Liver is producing Urea, but the kidneys are unable to filter it out that why
there is an elevation in BUN levels.
Reduced Blood Urea Nitrogen indicates your body isnt producing sufficient
amount of Urea! Because Urea only be produced in the liver that implies
LIVER DYSFUNCTION. Also elevated levels of transaminase could mean
Liver dysfunction.
Elevated levels of BUN generally will tell you its Kidney Dysfunction.
QUESTION: You are a test subject taking an investigational drug called
YMIinDisClass. Your blood test reveals reduced blood urea nitrogen (BUN)
and elevated levels of alanine transaminase (ALT). What can you conclude
based on this information?
A) Kidney dysfunction suggested by reduced area production in the kidneys
B) Kidney dysfunction suggested by elevated ALT
C) Liver dysfunction suggested by reduced BUN and elevated ALT
D) Brain dysfunction because I can't figure out the answer
Degradation of Cysteine, Alanine, Serine, Glycine, Threonine:
POINT YOU HAVE TO KNOW IS THAT ALANINE CAN BE
CONVERTED TO PYRUVATE!
Remember that Aspartate can be converted to Oxaloacetate
Know that asparagine is converted to aspartate! By L-Asparaginase which

generates free ammonia.
L-Asparaginase is important in Cancer Therapy, some patients are
administered this enzyme
Some cancer cells dont express Asparagine Sythetase(reverse of
Asparaginase) which converts Aspartate to Asparagine). Cancer cells are
dependent on Asparagine (for Building proteins and cell division itself)
SO IF YOU TREAT THE PATIENTS WITH ASPARAGINASE this will
REDUCE CIRCULATORY LEVELS OF Asparagine, the asparaginase
converts Asparagine to Aspartate and because cancer cells cant synthesize
Asparagine, there are susceptible and die off. Cancer cells dont have
Asparagine Synthetase. Some cancer cells cant synthesize Asparagine. You
introduce enzyme that breaks down Asparagine, you starve them and they
die. Example why basic pathways like this is very important.
REMEMBER: METHIONINE is broken down into SAM
(S-Adenosylmethionie)
Several Amino Acids are broken down to acetoacetate (a ketone body)
Degradation of Tryptophan to Acetoacetate(Ketone Body)
(Amino Acid to a-ketoacid)
Breakdown of Phenylalanine and Tyrosine:
Phenylalanine and Tyrosine metabolism is interesting. Inborn defects of
these enzymes associated with metabolic pathways.
People who defect in Phenylalanine Hydroxylase (they have Phenylalanine
in their urine). If they have a defect in this enzyme then they shouldnt take
Aspartate (can have phenylalanine in it) sweeteners.
Deficiencies in Fumarylacetoacetase have a buildup of Tyrosine! Main
point is that you can have many of these enzymes and have specific
pathologies associated with them.
Lecture 3: Purines and Pyrimidines Learning
Objectives:
Know the difference between Ribonucleotides and deoxyRibonucleotides.
Know the pathways for Purine and Pyrimidine Biosynthesis Know
the Basic structure of Pyrimidine Nucleotides.
Understand the role of TetraHydrofolate in Nucleotide Biosynthesis

Understand the basic pathway for nucleotide breakdown and its role in
human diseases.
Recording Notes: Kazochoa
1prime Carbon will be attached to the Nitrogenase Base. RNA will have
Uracil, DNA will have thymine. R-group in pictures stands for Ribose. 2
position in ribose can have a Hydroxyl Group or Hydrogen. If it has a
Hydroxyl group it will be a Ribose and it has Hydrogen it will be
deoxyribose.
3 position there is a Hydroxyl. 5 position you have a phosphate attached.
(One or more phosphates)
Important: A sugar with a base attached to it is called a Nucleoside!!
Now if there is a phosphate attached to the 5 end, it becomes nucleoside
monophosphate, if two phosphates it becomes Nucleoside diphosphate.
So anytime you have a base, Sugar, and any number of phosphates, it is
automatically called a NUCLEOTIDE!
Again you have five bases, Adenine,Guanine,Cytosine,Uracil,Thymine.
Why is Purine Nucleotides Important?
Nucleotides are important in Biosynthesis of DNA/RNA. You exploit
process of Biosynthesis of these nucleotides for therapeutic purposes.
ATP/GTP Energy Currency of the CELL!
To synthesize Purines it will come from a common precursor (IMP) Inosine
Monophosphate.
IMP is a special Xanthine Base (IMP), which has one phosphate (IMP will
be important when KARZAI discusses tRNA and protein biosynthesis and
Ribosome reaction).
Purine Biosynthesis:
Adenine and Guanine are not formed as free bases
Purine synthesis proceeds through a ribonucleotide intermediate (IMP)
Purine Nucleotides will be synthesize from a common precursor called IMP!

BIOSYNTHESIS OF IMP:
One of the reactions schemes to synthesize IMP. PRPP as a substrate, will
use Glutamine to donate an amine group to an l position. Glutamine is
important in this case as donor. Glutamine becomes Glutamate. This is
the example where he said that Glutamine is important in nucleotide
biosynthesis (IMP FORMATION)
Another Reaction involves THF (Again Methylation Reaction). In this it
donates a formyl group and donates it to another molecule, then after a
number of reactions you form IMP, which is the ULTIMATE precursor of
Adenine and Guanine nucleotides.
How Cells synthesize AMP or GMP from IMP?
So IMP has two fates, it can become AMP or GMP. This will depend on the
substrates that are available for the enzyme
So to form AMP you will requite Aspartate. (SHOULD KNOW) Aspartate
will donate one of its amines and form AMP.
Conversely IMP can be used to synthesize GMP and this will require
Glutamine (will donate an Amine group as well) SO
Aspartate for AMP
SO Glutamine for GMP
IMP DEHYDROGENASE: (Clinically Relevant) Important enzyme that
converts IMP to GMP
What could be a clinical indication for Drugs that block purine

biosynthesis?
Immunosuppressant during organ transplantation, Immunosuppressant used
to treat autoimmune disease such a rheumatoid arthritis and Crohns disease,
and chemotherapeutic drugs. So Immune cells (T-Cells will divide, when
they divide they have to synthesize DNA and RNA if you reduce purine
biosynthesis you will reduce Cell division.
Mycophenolic Acid: Is an inhibitor of IMP Dehydrogenase, again used as
an Immunosuppressant.
Thioguanine: Is an Inhibitor of IMP Dehydrogenase, used in remission
therapy for acute Non-lymphatic leukemias.
Not used during maintenance chemotherapy due to liver toxicity. Liver
toxicity develops following chronic use.
T&B Lymphocytes possess high levels of (IMP Dehydrogenase). Now we
formed AMP and GMP, we have to get it to the diphosphate form and
ultimately to Triphosphate forms.
Enzymes called Kinases (involve phosphorylation reactions)
Adenylate Kinase and Guanylate kinase will strip a molecule from ATP
and transfer it to GMP and AMP so it becomes ADP and GDP.
ADP and GDP going to triphosphate form is mediated by a single enzyme

(Very non-specific) reacts with other nucleotides as well. Like uracil and
thymine.
Nucleoside DiPhosphate Kinase: Will transfer a terminal phosphate from
ATP to a di-phosphate nucleoside. Nucleoside Diphosphate Kinase can use a
terminal phosphate from any triphosphate nucleotide.
Nucleoside Diphosphate Kinase is specifically governed by mass action.
Nonspecific enzyme that catalyzes the transfer of phosphoryl groups to XDP
to form XTP.
Nucleoside Diphosphate Kinase:
Non-specific enzyme that catalyzes the transfer of phosphoryl groups to XDP to form XTP.
Pyrimidine Synthesis: The Pyrimidine is Cytosine, Uracil, Thymine. In

essence why pyrimidines are handled differently. Pyrimidines are very
special, the difference between Uracil and Cytosine is the presence of an
Amine group this is IMPORTANT!
Pyrimidine Biosynthesis is very complex; we are going to start off with
UMP. (Uridine Monophosphate)
Cells have to have a mechanism to transfer it from UMP to UDP and UTP.
There are also kinases for Uracil. The specific one for UMP is Uridylate
Kinase converts UMP to UDP. Again you see Nucleoside diphosphate
kinase this converts UDP to UTP (again non-specific acts on all
Nucleotides). Now UTP can be converted to CTP (Only Triphosphate form).
Synthesis of CTP: Again the only difference between Uracil and Cytosine is
the presence of an amine group in cytosine. Glutamine will donate its side
chain to Uracil to convert it to Cytosine. By CTP synthetase(Requires ATP)
Again Glutamine is donating an amine group to uracil to form CTP.
Synthesis of CTP:
Question: Why do you think Uracil is not used in DNA? One good reason
is CTP can be converted back to UTP in the presence of oxygen or some
superoxide (oxygenated environment) the bond that keeps the Nitrogen in
Cytosine is weak and it is fairly easy to convert CTP to UTP. DNA repair
enzymes will recognize presence of Uracil and will excise it. If uracil was
there, you will get a quite a few excisions and many others.
Very Important enzyme that only acts on Ribo nucleotides in their
diphosphate form! The enzyme only acts on Ribonucleotides in their
diphosphate form. It converts a nucleotide NDP to dNDP(mean 2hydroxyl
group is missing)
For Ribonucleotide Reductase to work Nucleotide must be in diphosphate
form. So this Ribonucleotide Reducatase enzyme will act on ADP to form
dADP, UDP to form dUDP, CDP to form dCDP, GDP to form dGDP , and
CDP to form dCDP.
Formation of dNTPs(Same Non-Specific (Nucleoside diPhosphate Kinase)
will act on deoxynucleotides AS LONG ITS IN THE DIPHOSPHATE
FORM!
Formation of Thymine: Thymine is handled in a very special way;
conversion of Uracil to Thymine is handled by two different enzymes.
Difference between Uracil and Thymine is the presence of a methyl group in
Thymine going to involve one of the methyl donors.
What happens first is you form dUTP, but cells are unable to form Uracil that
is attached to deoxyribose, molecules that synthesizes DNA can incorporate
UTP in DNA (this is a PROBLEM) to prevent this cells use an enzyme
called dUTPase (an enzyme that cleaves dUTP very rapidly cleaves two
phosphates off dUTP. As soon as dUTP is formed it is rapidly converted to
dUMP(monophosphate).
Nucleotides in triphosphate form can be incorporated into DNA not in
monophosphate. This enzyme ensures that Uracil is not incorporated into
DNA so you form dUMP , and this dUMP will serve as a substrate for a
VERY IMPORTANT REACTION ( This is the Thymidylate Synthase
Reaction) this reaction will only involve the transfer of carbon. Transfer of
Carbon from N5,N10 Methylene
Tetrahydrofolate and adds it to Uracil to form Thymine. In this process you
also form oxidizes THF into dihydrofolate, which is very unusual this
thymidilate synthase enzyme will oxidize THF to dihydrofolate(this has
significance) .
OK SO WE START OFF with dUMP again we are methylated it to give
dTMP(which is done by Thymidylate Synthase) ,Carbon from N5,N10
Methylene THF is transferred to dUMP to form dTMP and in this process
N5,N10 Methylene is converted to DiHydrofolate . Then enzyme
DiHydrofolate Reductase will convert dihydrofolate back to
Tetrahydrofolate(THF) then THF can be charged again with a methyl group
from the first lecture with serine serving as the source(Serine donate carbon
to THF.)
Again Thymidylate Synthease converts dUMP to dTMP and
dihydrofolate reconverted back to Tetrahydrofolate.
Reason why this is important is because you can inhibit these enzymes.
For Example: IMP Dehydrogenase can be used therapeutically.
Step 2: Thymidylate Synthase:
Regeneration of Tetrahydrofolate:
Thymidylate Synthase which again converts dUMP to dTMP is targeted by

a number of drugs (FdUMP for example) again these drugs are called
Antimetabolites If you block Biosynthesis of TMP, with Thymidylate
synthase inhibitors, you reduce cancer cell proliferation.
Raltitrexed,(Used Currently) which is used for colorectal cancer, is a
Thymidylate synthase inhibitor
Dihydrofolate Reductase Inhibitors are called Antifolates
Ex. Methotrexate and Trimethoprim (targets bacterial dihydrofolate
reducatase) Dihydrofolate Reductase which converts DHF back to THF is
also targeted by a number of drugs.
Very Briefly discuss Nucleotide Breakdown: Uric Acid, direct metabolite
of Nucleotides. All broken down to Uric Acid. Point is there is a number of
Enzymes that convert AMP, IMP, XMP, GMP, to molecule called Xanthine
and it is the Xanthine, which becomes the substrate for an enzyme called
Xanthine Oxidase that converts (Xanthine to Uric Acid.) Ultimately
metabolizing nucleotides to Uric Acid. We excrete URIC ACID as we would
expect.
GOUT(Severe foot pain)
Which is characterize by an excess of Uric acid and reduced uric acid
excretion. This is treated with allopurinol which is an inhibitor of xanthine
oxidase(which converts xanthine to Uric Acid) Allopurinal is a pro-drug that
is converted to oxypurinol(in the liver) (Oxypurinol is what inhibits
Xanthine Oxidase.)
Another treatment for Gout is with a molecule called Probenecid (acts on
nephron) it inhibits reabsorption of uric acid and this will allow Uric acid to
be excreted!
Severe Combined Immunodeficiency:
Defects in the production of lymphocytes (T and B cells)
Individuals cannot mount an immune reaction against invading pathogens;
patients require a bone marrow transplant for survival. Reason why people
develop this condition is because there is a deficiency in nucleotide
catabolism. Specifically in breakdown of Purines.
Know that a cause of Severe Combined Immunodeficiency is Adenosine
deaminase deficiency (most common cause),will cause a buildup in
Adenosine nucleotides, Adenosine deaminase is abundantely expressed in
lymphocytes. Deficiency causes buildup of deoxyadenosine , which is toxic

to cells. Number of Reasons why Deoxyribonucleotdies are toxic.
Purine nucleoside phosphorylase deficiency (much rarer) results in buildup
of deoxyguanosine triphosphate, which is toxic to T lymphocytes! This
enzyme deficiency feedback and cause a buildup of metabolites
(UPSTREAM).
REMEMBER BUILDUP OF ADENSINE NUCLEOTIDES feeds back on
RIBONUCLEOTIDE REDUCTASE, which converts Adenosine to
deoxyadenosine causing the buildup of deoxyadenosine, which is toxic to
cells.
Like Targeting Cancer with Asparaginase.
Lecture 4: Nucleic Acids

Central dogma of molecular biology: Biological information flows from
DNA to RNA to protein! Ultimately its the structure of protein and to some
context the structure of RNA that is determining the function of a biological
process. It is the collected function of these different proteins inside the cell
that permit biological function on how organisms work.
DNAs ability to copy itself, how the structure of DNA is able to replicate
itself, the process that replicates Genomic information. Karzai will talk about
Transcription, transfer of information from DNA to RNA and translation
from RNA to protein. How when these functions are messed up they cause
diseases. Understand how Replication, Transcription and Translation are
regulated, what happens if they are mis-regulated.
IMPORTANT THEME IS HOW STRUCTURE IS RELATED TO
FUNCTION.
Nucleic Acids includes Deoxyribonucleic acid and Ribonucleic acid. A lot
of mRNA dont have specific structure, tRNA structure is very
unique(important in Translation) . 16srRNA in ribosome is a component of
the 30S ribosome.
Focus mainly on DNA structure and how it is related to its ability to
duplicate itself.
What is DNA?
Computers use 0,1 for their code, how is that code stored? It is stored in Bits,
you have magnets inside bits which can be positive or negative, that file is
stored in 0,1 code, that is stored on the hard drive.
0,1 of 3D printing is the A,T,C,G bases,
Computer file is the Gene, that is the file that describes the whole structure
of object.
Iron Oxide: Is the material that makes up the magnet in the HARD DRIVE.
DNA is the material, not yet the gene, DNA is JUST a chemical
(deoxyribonucleic Acid) That chemical is analogous to the magnet (makes
up Iron oxide). DNA is the material that stores the information. A, T (U),C,G
is the coding system.
3D plastic object (output) is analogous to the PROTEIN.
Computer file is the Gene Hard drive can be the genome.
The idea is that DNA is not the gene, code. DNA IS THE MATERIAL THAT
STORES A, T(U), C,G CODES ON IT. Make connections.
Learning Objectives for Part 1
Describes and interpret the experiments that showed DNA is the genetic
material? Explain why the structure of DNA suggests a copying mechanism?
What did Watson&Crick discover about DNA?
They built a model of the structure of DNA!
In the 1920s the questions were, what is the underlying mechanism for
heredity, the passing of trait from one parent to offspring. At that time people
knew chromosomes store hereditary information, however chromosomes
contain DNA and proteins. They didnt know(in 1920s) whether it was DNA or
protein that carried that information.
Two Sets of Experiments:
Fredericks Griffiths Experiment:(1928) A chemical resistant to heat is
responsible for the heredity if virulence in Streptococcus pneumonia.
Smooth Strain (Virulent) Has Sliming coat on it that gets attached to
mammalian cells infects mice and kills them.
Rough Strain (Non-Virulent) : Dont have slimy coat so mice will

probably live.
A chemical resistant to heat is responsible for the heredity of virulence in

Streptococcus pneumoniae
Frederick Griffith's Experiment (1928):
Does it prove DNA is the hereditary material for S. pneumoniae?

Griffiths did this experiment by first boiling smooth strain and breaking
apart smooth bacteria that are already viral and then injected it into mice.
(Mouse lives) YOU NEED LIVE SMOOTH BACTERIA TO KILL THE
CELL. But then he took Heat-killed smooth strain and rough strain (which
is live) and saw that the mouse died.
The conclusion from this Griffiths experiment is that there are some kind of
chemicals, that is resistant to heat and being picked up by the live rough
strain and transforming it to the Virulent form, that is why the mouse died.
CLEAR
Question: In The Frederick Griffiths Experiment, does it prove that DNA is
the hereditary material for S.pneumoria? No because with this boiled strain,
you actually have a soap of protein, DNA, RNA, and lipids that the Rough
Strain could potentially pick up to make themselves virulent, the Frederick
Griffiths experiment sensed that there is a chemical resistant to heat, and that
chemical is responsible for Virulence of heritance in S. pneumonia.
DNA IS RESPONSIBLE FOR THE HEREDITY OF VIRULENCE IN

STREPTOCOCCUS PNEUMONIAE
The Oswald Averys Experiment: First (Heat-killed smooth strain and
mixed with Rough Strain) Result the mouse dies.
Boiled strain (Heat-killed smooth strain) was treated with Protease first and
then mixes it with the ROUGH STRAIN.
Protease: Protein that will degrade all protein
Heat-killed smooth strain mixed with rough strain +Rnase Result: The
mouse dies! So you take boiled bacteria from smooth strain, you treat it with
Dnase which will chop up DNA, breakdown DNA into small pieces and mix
it with Rough Strain Result: IS THE MOUSE LIVES!
OSWALD AVERY EXPERIMENT, which showed that DNA is responsible
for the heredity of virulence in Streptococcus pneumoniae:
Oswald Avery's Experiment (1940s):
Avery basically took soup from boiled smooth strain and used purification
method to separate DNA from everything else, he mixed this DNA with
Rough bacteria, this experiment showed that if you degrade DNA, the Rough
strain cant pick this up material and therefore cant kill the mice. Once the
Rough strain picks up virulent DNA, they transform themselves into virulent
form, therefore kill the mice.
Because RNA is not responsible for Virulence, mice still die in the presence
of RNase. If you use Dnase, it will kill virulence and therefore mice live.
Must pre-treat bacteria to ensure that all bacterias cellular component is
degraded.
The next big Question: How does DNA pass on genetic information from
one generation to the next? What is the mechanism?
From one organism to its offspring
From cell to cell within the same organism. The answer lies within the DNA
structure.
Structure will be a major them throughout this course!!!
Watson and Crick only built a model of DNA that fit all the DATA. Their
model of DNA suggested that a copying mechanism.
The two strands of DNA have complementary sequences, when two strands
of DNA are separated, each strand can be used a template, for synthesis of
two new daughter strands.
By contrast the incorrect model proposed by Pauling and Corey did not
suggest a copying mechanism.
Learning Objectives for Part 11:
Describe the structure of B-Form DNA based on these characteristics:
Helical Sense, base-pairs per turn, rise per turn, diameter of the B-FORM
double helix, anti-parallel nature of the two strands, conformation of the
glycosyl bond(anti-or syn) and sugar pucker conformation.
Identify the major groove and minor grooves of DNA.
Draw the A=T and GC base pairs and the sugar phosphate backbone.
Explain the interactions that stabilize the DNA double helix.
Explain how DNA binding proteins could recognize specific DNA
sequences. B-FORM DNA is a right-handed double helix!
The Model of DNA Structure Proposed by Watson and Crick:
B-form DNA is a right-handed double helix
Diameter of DNA double helix is 20 ANGSTROMS or 20x10^-10 meters

There is a protein that helps DNA polymerase stay on the DNA. This
prevents polymerase from coming off (this protein is called B-Sliding
Clamp) or PCNA this protein helps polymerases stay on, has a hole that is
30Angstroms wide, you can immediately propose a mechanism on how
Bsliding clamp (or PCNA) helps protein stay on DNA it is basically a donut
that forms a ring around a DNA molecule and allows polymerase to stay on,
so knowing parameters is very important.
One base pair to the next is about 3.4 Angstroms wide. How
many base pairs are there in Humans?
3.2Billion Base pairs
How long is DNA when you stretch it out?
3x10^9bp x(3.4 x10^-10m)=10x10^-1m which is 1 meter! (Knowing this
number allows you to know how long is a piece of DNA when you stretch it
out from Human cell.
Number of Base pairs per turn is about 10.5bp per turn. Majority of DNA
inside cells is in the B-Form; B-form DNA is a right-handed double helix.
Directions your fingers are pointing are the direction you need to take up.
The building blocks of DNA and RNA are nucleotides.

A nucleotide is composed of three components: Base, a pentose sugar, and a
phosphate group!
Nucleoside: IS ONLY SUGAR AND BASE
Nucleoside Monophosphate is basically a nucleotide (which has one
phosphate sugar and a base.)
Responsible for numbering system in sugar ring.
How are nucleotides connected to each other?
Attached to each other through phosphor-diester bond. 3 end connected to
another phosphate (5 of another nucleotide) need to be able to describe
directionality. DNA is antiparallel to each other.
Lecture 5: DNA Structure and Replication

One Helical Turn: 10.5 base pairs
Anti-Conformation of Glycosyl Bond.

Base pointing away from the sugar ring. Two strands in order to pair
properly, the two strands must be in opposite directions, one 5to3 and other
5to3 in opposite direction. Pointing way from the ring that is the
anticonformation.
Glycosyl Bond: Is the bond between the sugar ring and the base.
Why isnt it in the Syn conformation?
Sugar-phosphate backbone is negatively charged, dont want to be close
together should be far apart. Therefore DNA likes to be in the
Anticonformation for the Glycosyl bond in the B-Form DNA.
Identifying Major and Minor Grooves:
First identify the sugar-phosphate backbone, then draw a line that is
tangential to curve of DNA(Parallel lines) and immediately you can see the
spacing. Use Jmol to see Electron density, can see grooves more easily
(Minor groove, which is narrower. Now if you go up on Major Groove it
doesnt cross over. Major Groove and Minor groove are separate.
Major Groove, Minor Groove, Sugar-phosphate backbone: This is what
molecules see sequences of DNA.
When you want to find a specific sequence, you use your eyes to look at
letters. Molecules SEE the structures of DNA, the molecule potentially
interacts with the sugar-phosphate backbone or insert itself in Minor Groove
or it will insert itself in the Major Groove to identify and distinguish one
sequence from another.
How do molecules distinguish between sequences? Three structures that
DNA Binding proteins could potentially recognize:
1.Sugar phosphate backbone
2.Minor Groove
3.Major Groove
Useful Tips:
When determining the directionality of a DNA strand identify the 5 and 3
and first. To identify the 5 and 3 focus on one pentose Ring and then
identify the 5and 3carbons. Then draw an arrow.
Important: For the 5 end there doesnt have to be phosphate (What is on
the 5end DOESNT matter) What Determines the Directionality is the
Sugar Ring!
Sometimes when you want to synthesize DNA for an experiment, you want a
free 3 Hydroxyl group, sometimes a phosphate group but it depends how
you committed you are about the structure.
Watson-Crick Base Pairing in DNA (or RNA) is the structural basic for
complementary association between two nucleic acid strands.
A pairs specifically with T (U) and G with C. Watson and Crick base pairing
is one kind of interaction that stabilizes the double helix. Another type of
force that is stabilizing DNA is the stacking of bases, which cause by
hydrophobic interactions between one base pair to the next. Aromatic Rings,
which have a lot of, pi electrons above and below the ring. Hydrophobic
environment kind of like an oil drop (Like oil droplets that like stick to each
other) Vinegar is Hydrophilic, oily droplets (Bases stacking on top each
other which is favorable.) Imagine a cage of water forming around DNA
bases
Base Stacking in Nucleic Acids:
Hydrophobic, Van deer Waals and electrostatic interactions favor the
alignment of bases in an aqueous solution or within a polynucleotide chain,
the un-stacked orientation is disfavored.
B-Form DNA is the most prominent in nature in physiological conditions,
under different conditions ,DNA can have A-DNA or Z-DNA.
A-DNA is more compact and shorter for same amount of Base pairs.
(RightHanded Helix)
Z-Form: Zig-Zig, Left handed helix maybe find Z-Form in alternating G/C
rich regions.
DNA (invivo) mostly found in B-Form DNA.
DNA Adopts Different Helical Forms:

RNA also can form Helices. RNA is more like the A-Form of DNA.
Sugar pucker conformation:
C-2ENDO vs. C-3ENDO
B-FORM: (C2 endo sugar pucker) sp3 carbons in sugar ring (angle of sp3
carbons is 109.5 ) Pentagon needs to give more angles to each carbon, so
every carbon has 109.5 angles.
In the C2 endo pucker the 2 carbon is higher than the 3carbon. In
the C3 endo pucker the 3 carbon is higher then 2 carbon.
Why is A-FORM more favorable for RNA?
Because in RNA you have 2Hydroxyl group that would interfere with the
C2Endo sugar pucker conformation (The 2 Hydroxyl group in RNA would
clash with the phosphate.) Thus why RNA is found predominantly in the C3
endo sugar pucker form (RNA preferentially in A FORM.)
RNA structure tends to be more diverse, the extra 2 Hydroxyl group in
RNA, which can form Hydrogen bonds with neighboring oxygen, or interact
with water molecule forming bridges (extra conformations) RNA structure is
more diverse.
How does a DNA BINDING PROTEIN (transcription factor) find a specific
DNA sequence?
If you were a DNA binding protein molecule, how would you find a
particular sequence? You would find it through the major groove. Why?
The complicity of a sequence presented in the major groove is quite

complex; molecule can be designed to recognize particular interactions of
the sequences such as Hydrogen Bond-donor and acceptors. The structural
complexity of the minor groove is less. Structural Complexity on the Minor
groove is less.
The protein that wants to find a specific sequence, most likely will find the
MAJOR GROOVE!
There are actually proteins that want to bind to all DNA sequences; this
protein will probably bind to the Minor groove or the sugar-phosphate
backbone. Major Exception:
TATA-BOX BINDING PROTEIN: Which is a first protein that will bind to
promoter and start transcription, that protein will insert itself in the Minor
groove.
How proteins recognize different species by recognizing major and minor
grooves.
Structural Motifs of DNA binding proteins
These molecules insert themselves in Major Groove
Helix-turn-Helix motif
Basic Helix-loop-helix motif
Basic Leucine Zipper Motif
Structural Motifs of DNA Binding Proteins:
Sugar Pucker Conformation: C-2' endo vs. C-3' endo:
Hydrogen-Bond Donor and Acceptor Atoms in the Major and Minor

grooves of DNA:
Hydrogen Bond donor and acceptor atoms in the Major and Minor Groove of
DNA. Notice how the four possible base pairs are chemically distinct in the
major and minor groove.
Lecture 6: DNA Structure and Replication
Learning Objectives of Part 2: Determine whether a DNA molecule is
relaxed or stressed based on the structural parameters of DNA.
Describe the process of DNA denaturation and annealing (or renaturation)
Explain why annealing two complementary DNA is a slow process
Describe what are hypo chromatic and hyper chromatic effects of DNA.
Interpret experimental data that measures DNA denaturation and annealing.
Notes from Recording:
It is possible to unwind DNA because six of the bonds in the nucleotides can
rotate freely, including the bond with the base. Bonds can rotate to allow
changes to DNA, therefore you can from a helical form to an unwound
parallel strands.
Question:
What is the number of base pairs per turn in relaxed dsDNA?

Relaxed dsDNA (B-FORM DNA in physiological conditions.) 10.5 base pair
per turn.
Unwound form is very unstable.
3.4 Angstroms are the distance from 1bp to the next.
Remember when something deviates from natural form of DNA its is going
to move back to the more stable form. It is like a spring and you want to
come back to state where number of base pairs per turn is 10.5.
How do you go from Natural double helical helix to completely separated
strands?
You will need to force double helix to unwind it. Unwind it to the point
where it almost becomes straight parallel. You need to put in energy one
form of energy is heat. Heat up DNA samples and separate into two strands,
this process is called DENATURATION! Or melting of DNA.
Denaturation is a rapid cooperative process. Moving ribbons around will
help denaturation of neighboring sequences, melting of one part of DNA will
help the melting of the Neighbor.
The melting temperature TM of a duplex DNA is the temperature at which
half of the DNA in the sample is denatured (Half is in double-helical form
and the other is unwound.
In the opposite direction going from two single-stranded DNA back to
anneal double strand this reverse process is called (Annealing) or
renaturation.
When you are heating up DNA at very high temperature like 95C and you
are slowly changing temperature back to room temperature, the DNA
sequences will find each other and then twist around each other to form
double-helical structure.
Opposite process is more complicated because sequences can be all over the
place, they need to find complementary sequences first and then pair up and
find each other, and the FINDING PART IS the slow process (Because you
need to look for the rite partner). But once the sequences find each other, the
forming of the double helix is a fast process!
If you go TOO QUICKLY from 95C to 25C , what will happen , you might
not find perfect matches, sometimes there will be sequences with the same
strand of DNA and you form these loops that are complementary to each
other and form these local structures and sometimes youll find sequences
that part of the sequence is complementary to a second molecule of the DNA

and form inter-strand secondary structure and single stranded regions.
So if you change too fast from 95C to 25C you have all sorts of structures
like Stem loop (intra-strand), (inter-strand secondary structure) or
singlestranded regions. That is why you need enough time for molecules to
find each other.
Genome Complexity: Means you have sequences like 100 molecules of a
dS DNA with same sequences versus 100 molecules dsDNA of different
sequences. The 100 molecules of different sequences of dsDNA will take
longer time to find each other.
Therefore when you have DNA sequences that are more complex, it will take
longer time for Renaturation process. Hrs vs. minutes.
Longer strand would take more time, when its longer it has a greater chance
of forming (intra-molecular structure).
If denatured DNA is not given enough time to find its complementary

strands during re-naturation, single-stranded DNA (ssDNA), stem-loops
and other inter- or intra-strand structures could form:
Heat can separate DNA, but also Increasing pH is also a method to denature
DNA the hydrogen bonds will become deprotonated, and cannot form
Hydrogen bonds. Why does this make it fall apart? The sugar phosphate
backbone is negatively charged and causes them to fall apart (-) charges
repel each other.
NOW if you add ACID to the DNA, the DNA will aggregate the sugar
phosphate backbone becomes less negative, therefore they stick each other.
How do you measure change in structure of DNA?
How do we actually detect molecules of DNA separate from each other, one
way is to look at the UV absorption of DNA. Visible light has a wavelength
of 400-700nm.UV has a wavelength much shorter 260nm; the pies electrons
above and below the bases absorb 260nm waves very well. When bases are
engaged in Hydrogen Bonding the probability of excited these electrons
become a lot lower. When bases are involved in Hydrogen bonding you
absorb LESS UV light. This is called the Hypo chromatic effect.(Less
color, Less UV light) .
Double-Stranded DNA involved in Hydrogen Bonding absorb less light, so
when you heat it up and separate two strands and bases no longer involved in
H-Bonding, therefore you absorb more light that process is called the
Hyperchromatic effect. You can measure this change in a cuvette holding
on to a DNA Sample, as you are increasing the temperature from room to
100 C you will see the absorbance is going to change from Low to High.
The reason it changes from low to high it because initially you have dsDNA,
as it dissociates it immediately changes to a high absorbance situation. The
midpoint between transitions is melting temperature because this is point
where half of molecule is still double-stranded and other half is single
stranded.
Which A260 Profiles Correspond to Which SNA Sequences:
DNA Replication: How do cells use one strand, two strands as templates to
synthesize the new daughter strands?
Describe the structure of the substrates of DNA replication
Describe the chemistry of DNA synthesis and the catalytic mechanism of
DNA polymerase.
Explain the molecular mechanism of DNA polymerase that ensures the
accuracy of the polymerase reaction
Explain how DNA polymerase reaction is primed.
Focus on chemistry of DNA synthesis and molecular mechanism of DNA
replication.
Chemistry of Reaction: Zooming in at atomic levels, trying to understand
how bonds are breaking, what electrons are attacking what.
Molecular Mechanism: Zoom out 1 step. Look at the Proteins! The
Chemistry of DNA Replication: the substrates of DNA Replication has two
substrates 1.) dNTPS.
2.) Primer/template junction: Two strands, a template strand have an
ssDNA and dsDNA. ssDNA will provide a template strand, directs the
synthesis of complementary nucleic acid.
The primer strand needs to have a 3OH group that will be used to extend the
strand. Primer Strand can be DNA or RNA. However the newly synthesized
product is DNA. Extend from the 3OH on the primer strand in the 5to 3
direction. DNA synthesis does NOT occur without a primer.
Remember you need a 3OH group to extend.

Overview of the Chemistry DNA Synthesis and the Molecular Mechanism
of DNA Replication:
The Chemistry of DNA Synthesis: Formation of phosphodiester Bond through

SN2 reaction.
So incoming dNTP, forms a Watson-Crick base pair, it will be position so the
phosphate will be very close to the Hydroxyl Group. 3OH will attack the alpha
phosphate of incoming dNTP in an SN2 reaction. SN2 reaction is coordinated;
OH group is the Nucleophile (loves Nucleus of phosphorous). The goal is to

form a phosphodiester bond, cleaving phosphorous oxygen bond, the leaving
group is pyrophosphate.
Nucleophile: 3OH on the primer strand attacks alpha-phosphate Leaving
Group: Pyrophosphate of the incoming dNTP.
When dNTP is not complementary to each other, the nucleotides arent lined
up properly.
Catalytic Efficiency: Is strongly dependent on the ability of the incoming
nucleotide to form complementary base pairing with the template.
PCR Reaction: Put in a DNA, and a primer, polymerase, dNTPS and then
you will synthesize product based on template strand.
When you mix DNA, template, and primer together with dNTPS NOTHING
will happen, without the enzyme (there is an Activation energy barrier), you
wont spontaneously go through this reaction it will take years. You need
enzyme to lower activation energy barrier. DNA polymerase is enzyme that
is lowering the energy. Incoming dNTP will form phosphodiester bond.
The Chemistry of DNA Synthesis: Formation of Phosphodiester Bond
Through SN 2 Reaction:
The active site of DNA polymerase can conceptually be separated into postinsertion site and insertion site. The post-insertion site is where its holding
already paired base pairs; it also holds nucleotide on primer-strand that contains
Hydroxyl Group, which is going to attack incoming dNTP.
Insertion site is where incoming nucleotide is going to fit in, it also contains
template that is complementary to incoming nucleotide. (dNTP) also place
where base pairing of new nucleotides is going to occur.
The Enzyme that Catalyzes the Synthesis of DNA is Called DNA
Polymerase (Pol):
Remember the POST INSERTION SITE is where attacking nucleotide is

located.
Insertion Site: Where incoming nucleotide dNTP also place where base
pairing of new nucleotide is going to occur.
DNA POLYMERASE 1
Molecule has a finger domain, thumb domain, palm domain. Thumb domain
grabs the double stranded region. Fingers are very flexible, could push in
dNTPs and palm domains contains ions (that help facilitate catalysis
reaction)
Active Site of DNA Polymerase: Lowers Activation Energy
DNA polymerase uses a two metal mechanism of catalysis. On the palm
domain of the enzyme it has amino acid residues Aspartate (that coordinate
two Mg ions) Negatively charged oxygens will put these Mg in place, place
them very close to attacking oxygen 3OH and also put it very close to the
phosphates of dNTP.
Why would this make the reaction better? Why would this make an SN2
reaction better?
Better leaving group, better nucleophile (get rid of H+) to make it 0- How
you make pyrophosphate a better leaving group (Neutralize negative
charge, make it less negative (Mg neutralizes some of the charge, thereby
making the pyrophosphate a better leaving group and Mg also stabilizes

attacking Hydroxyl Group to make it a better nucleophile.
Lecture 7:
Describe the structure of the substrates of DNA replication
Describe the chemistry of DNA synthesis and the catalytic mechanism of
DNA polymerase!
Explain the molecular mechanism of insertion fidelity (or accuracy) in the
polymerase reaction. How can enzyme ensure that correct bases are pairing
up with the template strand to allow for synthesis of correct complementary
DNA strands?
Recording: Under physiological conditions-FORM DNA is preferred
conformation (most relaxed).
What is the number of base pair per turn in relaxed dsDNA? 10.5bp
Clarification:
Over Wound DNA RelaxedDNA
UnderwoundDNA
Less then 10.5
10.5bp
Greater then 10.5
B-Form DNA that is relaxed form.

Overwound: Fewer Base pairs per turn, so this is overwound, remember
wants to be in relaxed stable form.
If you under wound you will have more base pairs per turn. This number will
be bigger then 10.5 base pairs per turn. This now explains the molecular
mechanism of insertion fidelity (accuracy) in the polymerase reaction.
How can the enzyme ensure correct bases are interacting with template
strand?
Watson-Crick said that energy from H-bonded is what is helping enzyme
find the rite nucleotides, however when people measure energy, with correct
matches, and then with incorrect mismatches, the energy difference with
match and mismatch is free energy -2kcal/mole, in theory an error rate of 1
in 100 insertions. But in reality when you do an experiment you find an error
rate for polymerase you find 1 in 100,000 insertions. The error rate of
polymerase-mediated insertion is 1 in 100,000. This corresponds to a
DELTA G of -7 kcal/mole between matched and mismatched base pairs.
DNA polymerases active site amplifies the free energy differences between
matched and mismatched base pairs (Base pair geometry and induced fit.)
Discusses how the enzyme is going to amplify energy difference between
matched and mismatched base.
If bases flip around CG and TA minor groove distance is similar.
When you analyze active site of DNA polymerase, when you have correct
base pair, sitting in active site of DNA polymerase, you find that it fits very
well in the active site.
By contrast if you have a mismatch base pair, you will find bases will stick
out, they clash into surface of the enzyme and they wont be able to form a go
od interaction with the surface of the enzyme. Emphasize when you have a
correct base pair that fits well in the active site, there are extra H-Bonds that
are interacting with minor groove of DNA (minor groove side of the base
pairs)
Palm domain H-bonding with the Minor groove, minor groove side of the
base pairs. Enzyme is stabilizing structure by forming extra H bonds.
Why Minor Groove?
Enzyme does want specificity to able to select correct nucleotide (Watson
and Crick Base Pair, but doesnt want so much specificity that will
distinguish AT and GC, the enzyme has evolved where it stabilizes bases
facing the minor groove. Enzyme needs similar efficiency for all bases, but
it must be able to distinguish wrong base pairs (so what its looking for is that
you can form (correct Watson and Crick Base Pairs.)These Hydrogen Bonds
provide that extra energy that you can distinguish correct base pair versus
not correct base pair.
Palm domain also interacts with the minor groove of correct nascent Base
pair, stabilizing the interaction. When you form bonds you form extra
stabilization energy. Energy with two H bonds and three H bonds, that
energy difference is only -2kcal/mole. That is not enough to distinguish rite
and wrong. The active site of DNA polymerases distinguishes rite and wrong
thus accounting for a bigger energy difference then -2kcal/mole.
The Second Molecular Basic for polymerase accuracy: Is the enzyme will
change conformation, undergoes a conformation change from an open form
to closed form. The thumb domain holds on to the double helical part of the
primer-template junction, the template is sticking out, finger domain is
flexible it can close, when nucleotides come in, it will bind to fingers and
move to the active site.
If you have a correct base nucleotide that can pair up with template-strand it
will be able to close completely. But if you dont have a correct base you
cannot close completely.
The finger domain of DNA Polymerase 1 consists of O-Helix. This O-Helix
is part of the finger domain. When incoming dNTP can form a good Watson
and crick base pair, then whole finger domain can close completely that also
increases the free-energy difference because you have extra interactions that
are provided by Lysine, Arginine, Tyrosine that is on surface of 0-Helix.
What is the charge characteristic of Lysine, Arginine? (+ Positive)
Can bind well with negatively charged phosphates group, in addition the
Tyrosine that is very hydrophobic is going to interact with incoming base
(the aromatic ring) this will add extra energy to tell difference between right
and wrong.
Can think of dNTP binding to the finger domain first and then closed up.
And if its not the correct base pairs, it wont close completely. This
nucleotide is coming to the active site and bp and thus closes completely to
stabilize the structure, when it is stabilize the Argine and lysine will further
stabilize the pyrophosphate leaving group, further distributes the negative
charge on pyrophosphate making it an even better leaving group, it also
stabilizes the stacking of the bases.
When its correctly base-paired with the complementary of the template

strand, this is the second basis on how the enzyme actually exaggerates the
energy difference between a mismatch and matched base.
Only when you get correct base pairing (Watson and Crick) will allow you to
have complete closing of the active site to allow the movement of finger
domain, thereby properly aligning the 3OH group and align it so it can
attack alpha phosphate to allow SN2 reaction to occur efficiently.
Incorrect Base pair, will not allow positioning of dNTP in active site
properly, this will decrease the rate of SN2 reaction by 10 fold to 1000 fold,
just MUCH slower reaction.
Remember everyone can go into the active site; all bases can go, however
only when correct nucleotides, dNTPs enter in the active site will have
COMPLETE CONFORMATION closing.
Can think of DNA polymerase as a nondiscrinative employer, he or she will
let anyone try out for the job, but only the one that can do the job will get.
Enzyme will let all four dNTPs come in to try, and only the one that works
will be allowed to go through an efficient SN2 reaction.
1# Two Mg ions, first is stabilizing the attacking 3OH group to make it
negative, becomes much stronger nucleophile and attacks the alpha
phosphate and second Mg that stabilizes leaving pyrophosphate and Argine
and Lysine in the finger domains will also stabilize the Pyrophosphate. The
Mg is on the palm domain and the finger domain; there are these residues
that can form H bonds through Minor groove of base pair. Tyrosine stabilizes
incoming Aromatic Ring of the incoming dNTP.
In nature some other DNA polymerases use other metal ions Mn,Mg,Zinc.
Polymerase Insertion Fidelity: Molecular Basic contributes to accuracy of
enzyme that allows it to make a mistake 1 in 100,000nts(nucleotides). This
isnt very good. Think about it if you have 3 Billion base pairs, that would
mean 30,000 mistakes! NOT GOOD ENOUGH!
In one round of DNA Replication. How can we make this better talk about
proofreading mechanism? Which involves 3 to 5 exonuclease activity of
enzyme, which will increase accuracy to one mistake in 10 million
nucleotides (Still NOT GOOD ENOUGH BUT PRETTY GOOD).
Explain the proofreading function mediated by the 3to5 exonuclease domain

of the DNA polymerase.
Explain how the 5to 3 exonuclease domain of DNA Pol 1 is involved in nick
translation. Describe what trans lesion DNA Polymerases. Describe how DNA
synthesis is primed.
Exonuclease cleaves nucleotides (at phosphodiester bond) one at a time from
the end of a DNA strand.
By contrast, endonuclease cuts the phosphodiester bond within a DNA
strand.
There are FIVE (5) DNA polymerases in Bacteria and a lot more in Humans,
basically the same.
These DNA polymerases also have extra domains that can do different
functions. DNA polymerases 1,2,3 have these 3to5 exonucleases, in Pol1
has this 5to 3 exonucleases.
Active site of DNA polymerase that is responsible for extending 3 end of
DNA and it has two other domains, its part of the same protein, translated as
one protein but just different domains on the enzyme. So it has the (5to3
DNA Polymerase, 3to5exonuclease, and 5to3 exonuclease).
It seems somewhat counterintuitive for a polymerase which syntheses in the

5 to 3 direction, to have 3to5 exonuclease activity. As it turns out, the
3to5 plays a critical role in ensuring the incorporation of correct nucleotide.
1 in 100,000 times you will incorporate wrong nucleotide, then you have
Non-Watson Crick base pairing, the enzyme might push it into the
postinsertion site, it might have a hard time moving into insertion site,
(Incorrect Nucleotide is out of register so you have all this steric hindrance)
and the nucleotide will flip it out and enter the 3to5 exonuclease domain
and that is the time it chews up that piece of DNA.
What is happening in the active site of exonuclease domain?

So you have an incorrect nucleotide entering, so it got stuck, it stalled there.
Gives opportunity for 3 to 4 nucleotides to flip out, and then flip it onto the
active site of 3to 5 exonuclease(remember its a different active site from
the DNA Polymerase youll find in the palm domain) , if you zoom in into
active site of 3 to 5 exonuclease you will once again see this 2-metal
mechanism.
Similar pattern to palm domain active site, this is essentially reverse reaction
of DNA synthesis, it uses Mg stabilizing 3Oxygen and another Mg
stabilizes (-) charge on the phosphate group and an incoming Hydroxyl
group.
The Nucleophile of this reaction (Hydroxyl Group is nucleophile, it is
attacking the nucleus of phosphorus its causing phosphorus oxygen bond to
break and what is the leaving group 3R0- (poor leaving group) so you have
a Mg close to it to distribute the charge (making it a better leaving group)
which helps the enzyme. Mg+ helps the deprotation of water. Hydroxyl
group that is acting as a Nucleophile is coming from the water(The
Magnesium helps the deprotonation. Magnesium makes the Hydroxyl group
a better Nucleophile.
When it is the correct dGTP, it will have perfect geometry fitting, and the
finger domains can close fully, facilitating SN2 reaction (will be very fast
with correct dNTP, forms phosphodiester bond and moves on to the next
cycle.
What happens when you have the incorrect nucleotide dNTP? Which will
happen 3 out 4 times
There is no correct geometric fitting; the finger domain cant close fully, so it
will just STALL THERE, the 3OH and alpha phosphate are not aligned
therefore reaction is extremely slow, it basically comes off, goes backward
However 1 in 100,000 times there will be a mismatched it can happen and
even if SN2 reaction is slow, the incorrect base pair that got incorporated,
now DNA will flip out and enter the exonuclease site, this is the time it will
get cleaved and go back to state.
But 1 in 10million times this will still happen it will force itself through and
keep on going and you will have a mismatch in the middle of the DNA
Sequence (this will be taken care of by DNA Repair which fixes
mismatches)
THOUGHT QUESTION: What happens if there is DNA damage on
template strand and destroys the ability to pair up with any dNTPs. Answer:
Paused Polymerase! Polymerase will just pause there and cant do anything.
Worst thing that can happen because you cannot complete the synthesis of
DNA, however cells have evolved Translesion DNA Polymerases.
What will happen? If you have a Gap who will give 3Hydroxyl Group, In
order to synthesize DNA you need 30H group to attack alpha phosphate, if
you have a gap there is no 3OH group, DNA can be locked in that
conformation. So you have a paused polymerase. Polymerase will be

paused and then it cant do anything (Worst thing that can happen to a cell,
because you cannot complete synthesis of DNA.). Cells
have evolved Translesions DNA Polymerase,
POL 4 AND 5 dont have 3to 5 exonucleases, these
enzymes have a much bigger cavity in the active site,
meaning that it can accommodate a lot of different
conformations.
What does it mean if you have a bigger cavity? It can work through it the
incorrect nucleotides might also be incorporated, Pol 4 and 5 have very high
error rates but are Essential to the cell because they help the cell go through
the process when you have paused polymerases.
Tranlesion DNA Polymerase: (Pol 4 and Pol 5) have low accuracy they
dont have 3to5 exonucleases or 5to 3 exonucleases.
The low accuracy of Translesion DNA polymerases allows them to insert an
incorrect nucleotide opposite to a damaged-template strand.
This allows the replication fork to move pass a damage site. Paused
polymerase happens very frequently, so you dont have want to undergo
apoptosis all the time.
Overview of the Mechanistic Basis for DNA Polymerase Accuracy:

Explain how the 5 to 3 Exonuclease domain of DNA Pol 1 is involved in
Nick Translation
Remember that the 5 to 3 exonuclease is ONLY found in polymerase 1. It is

involved in removing the primer used in DNA synthesis. The 5 to 3
exonuclease is sitting in front of the enzyme and moving forward if it
encounters a strand it will eat up DNA from the 5 to 3. In order to
understand why need this enzyme, you need to understand how DNA
Synthesis starts.
WHY DO YOU NEED THIS?
In order to appreciate 5to 3 exonuclease you need to understand how
DNA synthesis starts. Lets say double-stranded circle is E. Coli genomic
DNA, How do you start synthesis here? You probably melt out a part and
expose (two single-stranded templates) and use that template to synthesize.
DNA polymerase needs to have primer template junction, which means
you need to have properly annealed dsRegion with a 30H group (on both
strands and then polymerase can come in and extend in both directions)
Who is synthesizing small piece of RNA?
It needs an enzyme called primase, which is actually an RNA polymerase
which will use template sequence to synthesize a small piece of RNA, and
then will be recognize as a primer-template junction by DNA polymerase
and extend it to form these DNA strands. The small RNA primer is about 1113 nt long.
Important: DNA synthesis can be primed by RNA or DNA. In nature (in
vivo) you need to have short RNA to prime to start synthesis. However the
enzyme can bind a small piece of DNA and can start this how you do your
PCR Reaction can happen in Nature and the enzyme falls off and then comes
back on and has to continue synthesizing.
So once you have RNA incorporated into genomic DNA how do you remove
it? Why do you need to remove RNA? RNA is terrible genetic material,
because its not stable, it is unstable because it has that 2Hydroxyl Group
which is very close to the phosphate and can easily attack itself and cleave
itself.
RNA can easily degrade itself, and happens more frequently when in alkaline
conditions (will make the 2Hydroxyl group a better nucleophile.) RNA
Primase is also very error prone, system doesnt want to use RNA as a
storage material because it will incorporate lots of error. The enzyme will
remove it and to remove it, it will use 5 to 3 exonuclease and when Pol 1
encounters 5 end of the strand it will eat it up and replace it with DNA and
thats how it replaces RNA strand. This process is called Nick Translation.
A NICK is basically a gap between one strand and the other and that
nick is going to move UPWARD!

Slide: 5to 3 exonuclease of Pol 1 is involved in nick Translation: RNA is
unstable (not best for storing genetic information) RNA primase is also error
prone. Therefore RNA primer needs to be removed. The 5to 3 exonuclease
activity of Pol 1 removes the RNA(and DNA) in front of the polymerase.
This process is called Nick Translation, it important for removing the RNA
primers and DNA repair.
Nick Translation in POL1:
The 5 to 3 degrades RNA or DNA from the 5end. Simultaneously the
polymerase extends the 3end of the primer at the nick in the same
direction(5 to 3) By contrast Pol 2,3,4,5 cannot perform nick translation.
After Pol 1 falls off, the nick is sealed by DNA ligase!
Nick Translation by Pol 1:
Nick Translation by Pol 1: DNA polymerase 1 is organized into three major

domains.
1.) DNA Polymerase
2.) 3to 5 exonuclease activity
3.) 5 to 3 exonuclease activity
At a nick-here the gap between laggings strand fragments. Pol 1 degrades the
RNA primer in the 5 to 3 direction, releasing dNMPS and simultaneously
extends the 3 terminus with dNTPs in the same direction. The net result is
the movement of the nick in the 5 3 direction along the DNA until all RNA
is removed.
Concerted action of 5 to 3 excision and DNA polymerization is called Nick
Translation.
Nick Translation refers to the fact that a nick in the DNA gets translated
along the length of the strand.
Lecture 8: DNA REPLICATION, CONTINUED

Learning Objectives:
The difference between leading and lagging strands synthesis and the
molecular basis for these processes.
The difference between a processive and a distributive process.
How the B clamp increases the processivity of Pol 3 HOLOENYZYME.
(How B topologically links Pol 3 to the DNA.
How the B-clamp is loaded onto the primer-template junction.
The composition of the Pol 3 core and the Pol 3 Holoenyzme.
How the components of the replication machinery (Pol 3 core, B sliding
clamp y complex,helicase, primase,SSbs) are coordinated during replication
fork progression.
Lecture 8 Recording:
Remember primers can be DNA or RNA. Primer is a concept, where
polymerase is going to recognize to start DNA Replication. Primer is about
11-15 nucleotides long.
ENZYME PRIMASE is going to synthesize RNA fragments, which is 1015

nucleotides, which can be used as a primer for DNA Synthesis. Primers
arent always 10-15 nucleotides long. Primer is a concept, tells us where
DNA polymerase recognizes where it is complementary to the template, will
then extend at 3end to synthesize DNA. UNTIL 30H group is recognize as
a primer and starts extending.
Nick Translation: Is a process that can be catalyzed by DNA Polymerase 1
and the way it works. When DNA Pol 1 synthesizes DNA up to a point,
when it encounters something in front, it can be a piece of RNA or piece of
DNA, when it encounters that the 5 to 3 exonuclease kicks in and will start
chewing up whats in front, when it is chewing up whats in front it will open
up new template region for DNA Polymerase 1 activity sites to keep putting
new dNTPs ,incorporate nucleotides and as a result this position is moved
further up. (NOTHING TO DO PROTEIN TRANSLATION) it just means
Nick moved from one position to the other.
What is NICK? A NICK IS PAIRED UP ALL THE WAY UNTIL IT MEETS
THE END.
GAP: Has single-stranded DNA region that is not paired up.
Question: Why wouldnt Pol 1 keep going and fuse nick together?
You need to form phosphodiester bond, you need a Nucleoside
triphosphate, TRIPHOSPHATE IS GOING TO GIVE YOU THE ENERGY
YOU NEED TO MAKE THAT BOND. YOU NEED THE TRIPHOSPHATE
TO FORM PHOSPHODIESTER BOND. The Enzyme DNA Ligase seals the
NICK.
Replication Genome of E.Coli

He took E.Coli cells, broke it up gently, then spread genomic DNA on a
piece of membrane, these cells were previously grown in Radioactive
nucleotides, therefore genome incorporated into Radioactive nucleotides.
This genome is going to be a burn or mark when you look under the
microscope; he saw a lot of these kinds of structures.
Two Replication forks moving in opposite direction, they know DNA was
double helical, He always found that 1 and 2 were equal. DNA Replication
starts at single point, and it moves in both sides and the fork progressed and
makes it bigger and bigger until you get two full circles. In order for DNA to
be able to synthesize, both strands and knowing that DNA polymerase must
work in the 5to 3 direction, this implies, one strand needs to synthesize
continuously and other strand synthesize discontinuously. Also for DNA
polymerase to work, you need to prime it. (Lagging strand needs to be
primed frequently and not just once.
Okaziki discovered when you take genomic DNA from bacteria and separate
it on a gradient (a tube with solution (sucrose) which is heavier at bottom
and lighter at the top and you load genomic DNA and spin it, heavier
material to bottom and light material at the top, they found that they were
always these smaller pieces of DNA at lighter fractions of these gradients.
THIS SUGGESTS THAT AT LEAST ONE STRAND IS SYNTHESIZE
DISCONTINOUSLY.
Enzymes responsible for genomic DNA of E.Coli, one more player that is
DNA polymerase III. So DNA polymerase 1 was first identified because it
turns out that DNA Pol 1 is the most abundant DNA polymerase inside the
cell. But DNA polymerase 1 enzyme turns out to be extremely slows. If you
measure how many nucleotides it is going to add into growing template, it
turns out to be 20 nucleotides/per second.
So if E.Coli Genome is 5 million, so if you are synthezing DNA, two sets of

polymerases it means that one side (2.5million) . How long does it take to
synthesize that much DNA?
2.5 million /20(bp/s) gives you about 125,000 seconds to synthesize to 2.5
million base pairs or about 2000 minutes to synthesize a whole genome.
VERY LONG TIME! Doubling time of Bacteria is about 15-20 minutes.
A Mutation in polymerase activity of POL 1(The cells are still Viable, that
lets people use that strain to look for second polymerase that is in the cell
and that is responsible for replicating the entire genome, that lead to the
identification of Pol 2 and Pol 3, it turns out that DNA Pol 3 is responsible
for synthesis of leading and lagging strand. And this enzyme is much faster,
and it is the main replicator of the E.Coli genome also has a (VERY HIGH
PROCESSIVITY).
Pol 1 and Pol 3 BOTH HAVE 3TO 5 exonuclease activity remember this is
for proofreading.
But DNA Polymerase 1 is the only one that has the 5 to 3 exonuclease.
What happens if you mutate (5to 3 exonuclease)?
5 to 3 Exonuclease function is ESSENTIAL!
It turns out that the cell is NOT viable. Turns out that the function of Pol 1 is
to remove the RNA primer (fill in gaps, which is important for DNA repair
process. Removal of RNA primer is important for Lagging Strand. POL 3
is for Synthesis of LEADING AND LAGGING STAND. (Very
Important).
What is Processivity?
By definition it is the number of Nucleotides added to the growing primer
strand per binding event.
Non-Processive ( Distributive): Polymerase binds to the primer-template
junction , binds 1 dNTP ,add dNTP, forms 1 phosphodiester bond and then it
leaves. And the cycle goes back again and binds again. A completely
Nonprocessive process. By contrast a processive process, enzyme binds
primertemplate junction and then it keeps adding dNTPs to form long strand.
In theory fully processive process will keep going until it reaches the end.
Processivity of Pol 1 is about 10-100nt, means it will bind at 10 and then
come off, when it comes off it exposes an end. And Pol 3 core has an even
longer processivity BY ITSELF (1-10nucleotides)
The Pol 3 Holo-enzyme, which has a few extra subunits, is going to increase
processivity (to approximately 100,000 ntd per binding event) this increase
in processvity is due to a protein called B sliding clamp.
How does the Beta-Subunit (B-Clamp) improve the processivity of POL 3?

It turns out that when people isolate B-sliding Clamp, purified it and solved
crystal structure of B-Sliding Clamp, they found it looks like a Ring
Structure, when you measure the distance it is about 30Angstroms, which is
big enough to accommodate double-stranded DNA helix, and even thick
enough to accommodate a layer of water molecules, surrounding that piece
of double-stranded DNA, the layer of water is sort of lubricating B-Sliding
Clamp.
Question: How does the B-Sliding Clamp assemble onto the DNA in the
first place?
B-Sliding Clamp is composed of two subunits of same protein, so its called
a dimer. In Humans and Eukaryotes it has a very similar protein called
PCNA. (Proliferating Cell Nuclear Antigen) However, it is a trimeric protein
in Humans, and a dimer in E.Coli.
If this is such a nice circle, how does it assemble onto the DNA first place?
How B-Clamp helps polymerase stay attached to DNA and increase
processivity.
Distributive Synthesis: Where Pol 3 is binding to primer-template junction
extending a few nucleotides and then it falls off. But if B Clamp is present,
POL 3 will extend but even if it falls off, the B-Clamp is going to hold on to
it and keep it close to the primer-template junction, and let it resemble and
keep going, it doesnt mean it will keep it there all the time, but the B-clamp
will keep it around long enough, that it will bind to the active site to increase
processivity.
How does B-Clamp load onto primer-template junction turns out you need a
second protein called a gamma complex, which is an ATP-dependent process
complex. Gamma complex have five subunits, regions will bind to B-Clamp
when ATP is around, when ATP binds to gamma-complex it will cause a
conformational change in the gamma complex, in the gamma complex it will
move itself so it exposes binding to Beta, when it Binds, Beta it will open it
up and put a little crack, and give space for primertemplate junction to enter,
when it enters it is going to stimulate ATPase activity and Hydrolyze ATP to
ADP, when gamma complex bound to ADP, this protein will no longer have
affinity for Beta clamp , it comes off and when gamma complex comes off,
B-clamp closes itself, therefore its gets assemble onto primer-template
junction. Once you have that, you can attract Pol 3 to bind and extend that
region.
DNA Pol 3 (Holoenzyme) it has a few more components, you have two DNA
polymerase 3 core,Pol 3 core is like Pol 1 polymerase activity. Pol 3 can
bind to a Beta clamp, and there are two sets of these things, and there are
two sets of these things, y-complex is linked together by a subunit called 0).
B-Sliding Clamp attached to Pol 3.
DNA Pol 3 HoloEnzyme the main replicase of the E.Coli chromosome.
Pol 3 Core (x2)
Beta Sliding Clamp (x2) Each has two identical subunits(HOMODIMER).
E(3to 5 exonuclease)
Y Complex: Beta Clamp Loader
DNA Replication in the Context of the Chromosome:
As Pol 3 is moving along further up, Pol 3 will fall off, Pol 3 DOES not have
5to3 exonuclease. It gets to a certain point and then falls off.
Where is Pol 1 mediated DNA Synthesis taking place? Its taking place at
F, so it turns out that after DNA Pol 3 falls off, B-Sliding Clamp stays there
and the B-Sliding clamp is important for recruiting DNA pol 1, that helps it
find that RNA primer that needs to be removed.
Why would Pol 3 fall off?
Insertion site where dNTP needs to come in, but now 2Hydroxyl Group
basically crash into that site, finger domain which has that 0-Helix, it cannot
close, so now the Pol 3 enzyme falls off, it leaves a NICK NOT a GAP.
Summary:
The chemistry and Catalysis of DNA Synthesis are exactly the same at the
lagging strand and the leading strand.
However the leading strand is synthesized continuously, while the lagging
strand is synthesized discontinuously.
This is because the movement of the replication fork is opposite to lagging
strand synthesis.
Therefore, lagging strand synthesis must restart many times to fill in the
gaps.
Switching from Pol III to Pol I to Ligase:
IMPORTANT: So Pol 3 synthesizing, and when it gets to a point, it hits the

RNA primer from the previous synthesis and then it falls off then it will
leave behind the Beta clamp, and that Beta clamp is recognized by Pol 1, the
beta clamp doesnt help increase its processivity. Beta clamp helps recruit
Pol 1 there and Pol 1 can now fix RNA by using POL 1s 5 to 3
exonuclease, remove RNA and fill in with new DNA.
Why would DNA Pol 1 not keep going?
POL 1 CANNOT JOIN strands together (REALLY IMPORTANT), Pol 1
will eventually fall off because of its low processivity.
Remember those numbers what
PROCESSIVITY? 100,000 nucleotides
is
POL
HOLOENZYME
What is processivity of Pol 1? 10-100 nucleotides, remember POL 1

doesnt fuse two strands together.
Who fusing two strands together? DNA LIGASE!
Relaxed State of DNA has about 10.5 base pairs per turn. In physiological
conditions, B-Form DNA is predominant form. In solution people have not
seen (A form DNA) not a natural conformation for DNA.
Beta Clamp is behind Pol 3. Gamma complex is holding two pol threes to
one another. Pol 3 is a prokaryotic DNA polymerase. DnB will be on
lagging strand.
Primase: Guy that is synthesizing RNA Primer. Primase has some

interaction with Dnb, which tells it where to lay down RNA primer. Beta
clamp can also be found where there is a previous Okazaki fragment. Beta
clamp can also be found at the Gamma complex , when its with the Gamma
complex it is open, it is waiting to interact with the primer-template junction.
What the 5 to 3 exonuclease is doing is that it is cleaving the
phosphordiester bond, two metals ions will be very close to phosphodiester
bond, water is a nucleophile, but it is deprotonated because the metal ion
helps stabilizing negative charge water molecule, leaving group is this
strand, if it is 5 to3 exonuclease it will cleave the other way in which the 3
to 5 exonuclease works.
When you add acid sugar-phosphate backbone becomes protonated,
doublestranded region will interact with each other and aggregate. If pol 3 is
processive and Pol 3 keeps binding this site, where Pol 3 falls off, you will
give a chance for Pol 1 tom come in. If Pol 3 already sitting on strand, will
not get a chance for Pol 1 to come in. DNA POL 1 is so predominant; if you
expose 3 primer sites it is going to work on it.

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BIO 362 Exam 1 Review of Lecture Transcribed and Learning

Slide: How Does Ammonia become incorporated into Amino Acids?

Emphasized! MTHFR Reaction.

MTHFR MUTATIONS lead to elevated levels of HOMOCYSTEINE

GABA and Histamine:

Lecture 2: Amino Acid Degradation

Understand how the Urea cycle contributes to nitrogen elimination

Glutamine itself can be used as a source of Nitrogen, such as in Asparagine

Large subset of cellular proteins are broken down through

Massive Complex called the Proteasome(Has multiple proteins associated

Urea is predominant Nitrogenous species in Urine. Urea is the breakdown

Amino Acid Deamination: Deamination reactions are carried out by

generate free ammonia and regenerate a-ketoglurate. a-ketoglutarate to

Urea Cycle disorders: Treatments

Know that asparagine is converted to aspartate! By L-Asparaginase which

Understand the role of TetraHydrofolate in Nucleotide Biosynthesis

Purine Nucleotides will be synthesize from a common precursor called IMP!

What could be a clinical indication for Drugs that block purine

ADP and GDP going to triphosphate form is mediated by a single enzyme

Pyrimidine Synthesis: The Pyrimidine is Cytosine, Uracil, Thymine. In

Thymidylate Synthase which again converts dUMP to dTMP is targeted by

lymphocytes. Deficiency causes buildup of deoxyadenosine , which is toxic

Lecture 4: Nucleic Acids

Rough Strain (Non-Virulent) : Dont have slimy coat so mice will

A chemical resistant to heat is responsible for the heredity of virulence in

Does it prove DNA is the hereditary material for S. pneumoniae?

DNA IS RESPONSIBLE FOR THE HEREDITY OF VIRULENCE IN

Diameter of DNA double helix is 20 ANGSTROMS or 20x10^-10 meters

The building blocks of DNA and RNA are nucleotides.

Lecture 5: DNA Structure and Replication

Anti-Conformation of Glycosyl Bond.

DNA Adopts Different Helical Forms:

The complicity of a sequence presented in the major groove is quite

Sugar Pucker Conformation: C-2' endo vs. C-3' endo:

Hydrogen-Bond Donor and Acceptor Atoms in the Major and Minor

What is the number of base pairs per turn in relaxed dsDNA?

that part of the sequence is complementary to a second molecule of the DNA

If denatured DNA is not given enough time to find its complementary

Remember you need a 3OH group to extend.

The Chemistry of DNA Synthesis: Formation of phosphodiester Bond through

OH group is the Nucleophile (loves Nucleus of phosphorous). The goal is to

Remember the POST INSERTION SITE is where attacking nucleotide is

making the pyrophosphate a better leaving group and Mg also stabilizes

Less then 10.5

Greater then 10.5

B-Form DNA that is relaxed form.

When its correctly base-paired with the complementary of the template

Explain the proofreading function mediated by the 3to5 exonuclease domain

It seems somewhat counterintuitive for a polymerase which syntheses in the

What is happening in the active site of exonuclease domain?

conformation. So you have a paused polymerase. Polymerase will be

Learning Objectives for Part 3:

Remember that the 5 to 3 exonuclease is ONLY found in polymerase 1. It is

nick is going to move UPWARD!

Nick Translation by Pol 1:

Nick Translation by Pol 1: DNA polymerase 1 is organized into three major

Lecture 8: DNA REPLICATION, CONTINUED

ENZYME PRIMASE is going to synthesize RNA fragments, which is 1015

Replication Genome of E.Coli

So if E.Coli Genome is 5 million, so if you are synthezing DNA, two sets of

How does the Beta-Subunit (B-Clamp) improve the processivity of POL 3?

DNA Replication in the Context of the Chromosome:

Switching from Pol III to Pol I to Ligase:

IMPORTANT: So Pol 3 synthesizing, and when it gets to a point, it hits the

What is processivity of Pol 1? 10-100 nucleotides, remember POL 1

Primase: Guy that is synthesizing RNA Primer. Primase has some