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Important roles of nucleotides in cells Precursors of DNA and RNA Essential carriers of chemical energya role primarily of ATP and to some extent GTP Components of the cofactors NAD, FAD, S-adenosylmethionine, and coenzyme A
Some intermediates cAMP and cGMP, are also cellular second messengers. Two types of pathways lead to nucleotide synthesis: i) ii) the de novo pathways and the salvage pathways
de novo pathways
de novo synthesis of purine and pyrimidine - almost identical in all living organisms guanine, adenine, thymine, cytidine, and uracil - not intermediates in these pathways (the bases are not synthesized and then attached to ribose) The purine ring structure is built up one or a few atoms at a time and attached to ribose throughout the process The pyrimidine ring is synthesized as orotate, attached to ribose phosphate, and then converted to the common pyrimidine nucleotides required in nucleic acid synthesis.
Although the free bases are not intermediates in the de novo pathways, they are intermediates in some of the salvage pathways
Important precursors shared by the de novo pathways for synthesis of purines and pyrimidines: 1. Phosphoribosyl pyrophosphate (PRPP) 2. An amino acid glyc & asp.for purine and asp. for pyrimidine 3. Glutamine source for amide group in both the bases
Origin of the carbon and nitrogen atoms of the purine ring system
Stepwise description:
1. de novo purine synthesis begins with PRPP Committed step of the pathway a amino group donated by glutamine is attached at C-1 of PRPP resulting in 5- phosphoribosylamine highly unstable. Purine ring subsequently built up on this structure
2.
An ATP is consumed to activate the glycine carboxyl group (product- Glycinamide ribonucleotide) 3. Added glycine amino group formylated by formyltetrahydrofolate (product formylglycinamide ribonucleotide) N10
4.
A nitrogen is contributed by glutamine (product formylglycinamide ribonucleotide (FGAM) Dehydration and ring closure- Five-membered imidazole ring of the purine nucleus (product 5aminoimidazole ribonucleotide (AIR) Three of the six atoms needed for the second ring in the purine structure are present
5.
11. Second ring closure takes place to yield the second fused ring of the purine nucleus (product inosinate)
Conversion adenylate
of
Inosinate
to
Insertion of an amino group derived from aspartate takes place in two reactions similar to those used to introduce N-1 of the purine ring (addition of aspartate in one step and removal of fumerate in the next ). Difference is that GTP rather than ATP is the source of the highenergy phosphate in synthesizing adenylosuccinate Inosinate to guanylate Oxidation of inosinate at C-2, followed by Addition of an amino group derived from glutamine. ATP is cleaved to AMP and PPi in the final step
By removal of water from N-carbamoylaspartate the pyrimidine ring is closed to form L-dihydroorotate (dihydroorotase)
L-dihydroorotate is oxidized to the pyrimidine derivative orotate NAD is the ultimate electron acceptor (dihydroorotase dehydrogenase) PRPP again provides a side chain attached to orotate to yield orotidylate (orotate phosphoribosyl transferase) Orotidylate decarboxylase) decarboxylated to uridylate (orotidylate
Uridylate phosphorylated to UTP (ATP Kinases) CTP is formed from UTP by the action of cytidylate synthetase, one ATP consumed Glutamine the nitrogen donor.
dTMP
Largely through aspartate transcarbamoylase (ATCase) (catalyzing the first reaction in the pyrimidine synthesis Also inhibited by CTP, the end product of the sequence The bacterial ATCase : Consists of six catalytic subunits & and six regulatory subunits Catalytic subunits - bind the substrate molecules,
RPC
CPC
CPC
Allosteric subunits - bind the allosteric inhibitor, CTP. When CTP is not bound to the regulatory subunits maximally active. If CTP accumulates and binds to the regulatory subunits - undergo a change in conformation - the change is transmitted to the catalytic subunits, which then also shift to an inactive conformation.
RPC
Recycling of purine bases: Free purines are in large part salvaged (saved) and reused to make nucleotides, Salvage pathway - much simpler than the de novo synthesis of purine nucleotides. Uric acid is the excreted end product of purine catabolism in primates, birds, and some other animals One of the primary salvage pathways consists of a single reaction catalyzed by adenosine phosphoribosyltransferase, in which free adenine reacts with PRPP to yield the corresponding adenine nucleotide Adenine + PRPP AMP + PPi
to
lack
of
hypoxanthine-guanine
A lack of hypoxanthine-guanine phosphoribosyltransferase exclusively in male children, results in a set of symptoms called Lesch-Nyhan syndrome. becomes manifest by the age of 2 years, are sometimes poorly coordinated and mentally retarded. In addition, they are extremely hostile (unfriendly) and show compulsive selfdestructive tendencies: They mutilate themselves by biting off their fingers, toes, and lips. The devastating effects of Lesch-Nyhan syndrome illustrate the importance of the salvage pathways. Hypoxanthine and guanine arise constantly from the breakdown of nucleic acids. In the absence of hypoxanthineguanine phosphoribosyltransferase, PRPP levels rise and purines are overproduced by the de novo pathway, resulting in high levels of uric acid production and goutlike damage to tissue The brain is especially dependent on the salvage pathways, and this may account for the central nervous system damage in children with Lesch-Nyhan syndrome.
The pathways for degradation of pyrimidines generally lead to NH4 production and thus to urea synthesis. Thymine, for example, is degraded to methylmalonylsemialdehyde (Fig), an intermediate of valine catabolism. It is further degraded through propionyl-CoA and methylmalonyl-CoA to succinylCoA.
In 1971, Lehman IR discovered exonuclease I in E.coli Since then numerous exonuclease discovered: exonuclease, II, III, IV, V, VI, VII, and VIII Each type of exonuclease has a specific type of function or requirement. Exonuclease I breaks apart single-stranded DNA in a 3'=>5' direction, releasing deoxyribonucleoside 5'-monophosphates one after another. It does not cleave DNA strands with terminal 3'-OH groups because they are blocked by phosphoryl or acetyl groups. Exonuclease II is associated with DNA polymerase I, which contains a 5' exonuclease that clips off the RNA primer contained immediately upstream from the site of DNA synthesis in a 5' 3' manner. Exonuclease III has four catalytic activities:
3 to 5 exodeoxyribonuclease activity, which is specific for double-stranded DNA RNase activity 3 phosphate activity AP endonuclease activity (later found to be called endonuclease II)(AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA that has neither a purine nor a pyrimidine base, usually due to DNA damage Exonuclease IV adds a water molecule, so it can break the bond of an oligonucleotide to nucleoside 5 monophosphate. This exonuclease requires Mg2+ in order to function and works at higher temperatures than exonuclease I
Exonuclease V is a 3 to 5 hydrolyzing enzyme that catalyzes linear double-stranded DNA and single-stranded DNA, which requires Ca2+ Exonuclease VIII is 5 to 3 dimeric protein that does not require ATP or any gaps or nicks in the strand, but requires a free 5 OH group to carry out its function.
3' -->5' Activity 1. Removal of nucleotides (hydrolysis of DNA) takes place in the 3'-->5' direction.
5'-->3' Activity l. Hydrolysis is in the 5'-->3' direction. 2. Hydrolysis begins at the terminal phosphodiester bond or at a bond several residues away from the 5' end 3. Cleavage takes place irrespective of whether the 5' end is 5'hydroxyl or 5'-phosphorus. 4. The products may be 5' -mono- nucleotides, dinucleotides or oligonucleotides. 5. The cleaved bond must be in the double stranded region 6. The small fragment (MW 36,000) of polymerase I has 5'-->3' activity.
7. Has a role in the excision of pyrimidine dimers during repair replication. (The dimers are formed when DNA is exposed to UV radiation).
8. Polymerase I shows 5'-+3' exonuclease activity. This activity is reported to be absent in Pol 11 and Pol IIV although some works indicate that it may be present in Pol III
Restriction enzymes
Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA technology is totally dependent on restriction enzymes. Restriction enzymes are endonucleases
Bacterial enzymes
Different bacterial strains express different restriction enzymes The names of restriction enzymes are derived from the name of the bacterial strain they are isolated from Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragments Basic tools of gene cloning Names of restriction endonucleases Titles of restriction enzymes are derived from the first letter the first two letters of the species of organism from which they were isolated. EcoRI BamHI HindII PstI Sau3AI AvaI from Escherichiacoli from Bacillus amyloliquefaciens from Haemophilus influenzae from Providencia stuartii from Staphylococcus aureus from Anabaena variabilis of the genus +
Restriction enzymes recognize a specific short nucleotide sequence known as a restriction sites
Only type II restriction endonucleases are used for restriction mapping and gene cloning.
Palindrome: sequence of DNA that is the same when one strand is read from left to right or the other strand is read from right to left consists of adjacent inverted repeats
Enzyme
Organism from which derived Anabaena variabilis Bacillus amyloliquefaciens Bacillus globigii Escherichia coli RY 13
Eco RII
Hae III Hha I Hind III Hpa I Kpn I Mbo I Mbo I Pst I Sma I SstI Sal I Taq I Xma I
* C C A/T G G
GG*CC GCG*C A* A G C T T G T T * AA C G GTAC * C *G A T C *G A T C CTG CA* G CCC*GGG GAG CT* C G *TC GAC T* C GA C*CCGGG
Recognition site/sequence
Overhangs
Non-blunt ends overhangs. are created by various
The nucleotide sequence in duplex deoxyribonucleic acid (DNA) to which a restriction endonuclease binds initially and within which the endonuclease cuts the DNA.
Blunt ends The simplest DNA end of a double stranded molecule both strands terminate in a base pair not always desired in biotechnology
An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic. The simplest case of an overhang is a single nucleotide. This is most often adenosine and is created as a 3' overhang by some DNA polymerase.
when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends
When performing subcloning (moving a particular gene of interest from one vecor to another), it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired 5'-CTGATCTGACTGATGCGTATGCTAGT-3 3'-GACTAGACTGACTACGCATACGATCA-5'
Most commonly this is used in cloning PCR products created by such an enzyme. The product is joined with a linear DNA molecule with 3' thymine overhangs.
Since adenine and thymine form a base pair, this facilitates the joining of the two molecules by a ligase, yielding a circular molecule.
5'-ATCTGACTA-3' 3'-TAGACTGA-5'
Blunt end
Sticky end
Frayed ends refers to a region of a double stranded (or other multi-stranded) DNA molecule near the end with a significant proportion of non-complementary sequences; that is, a sequence where nucleotides on the adjacent strands do not match up correctly
5'-ATCTGACTAGGCA-3' 3'-TAGACTGACTACG-5'
non-complementary sequences are also possible in the middle of double stranded DNA But mismatched regions away from the ends are not referred to as "frayed".