Hanan

Enzymology
Nabeel of 20
Al-Fayomi 7
DNA
Basheer
13/10/2
replication
010

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Molecular Genetics – Lecture 7
Wednesday, October 13, 2010
Done by: Hanan Al-Fayomi

Before starting I would like to thank Suha Aqaileh, Razan Naji Ali
and Ahlam Zawaideh for helping me in this work.

Section 2: Enzymology of DNA replication

We’ll continue talking about enzymology of DNA replication.

 [slide 31]:

This is the slide I talked about last time explaining the different
types of DNA polymerase in prokaryotic and you saw last time that
we have polymerase I, II, III and we said that the principle
enzyme for DNA replication is polymerase III while DNA
polymerase I and II, especially I, are responsible for proofreading
and editing.

You noticed, also, that some enzymes have different functions, so they
are multi-functional enzymes.

For example, DNA pol I has three activities:

1) 5’----- 3’ polymerase activity

2) 5’-----3’ exonuclease activity

3) 3’----5’ exonuclease activity

 [slide 32] :structure of DNA polymerase I

This is a diagram for the tertiary structure of DNA pol I. Again,

DNA pol I is responsible for proofreading and filling the gaps
repairing DNA damage.

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 Which gaps are we talking about?
Gaps that result from the removal of RNA-primers used to initiate
the replication process.

 [slide 33]: Klenow fragment

As we said DNA pol I is a multi-functional enzyme. If we subject

DNA pol I to a proteolytic enzyme, it will degrade DNA pol I into
two fragments:

- Small fragment consists of 323 amino acids having 5´→3´

exonuclease activity.

- Large fragment, called klenow fragment, consists of 604

amino acid having

5’----3’ polymerase activity and 3’---5’ exonuclease activity.

 [slide 34]: DNA polymerase II

DNA polymerase II works once we don’t have or we have

nonfunctional DNA pol I and DNA pol III. DNA pol II participates
in DNA repairing mechanisms.

 [slide 35]: DNA polymerase III

DNA polymerase III is the principle enzyme for DNA elongation

and synthesis.

 [slide 36]: Structure of DNA pol III

This is the structure of DNA pol III which is a multi-subunit

enzyme and these are the principle subunit:

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 • α (alpha) subunits are responsible for elongation (polymerizing
activity).

• ε (epsilon) subunits are important for the 3’----5’ exonuclease

activity.

• θ (theta) subunits are important to assemble the enzyme structure.

• Also there are some accessory subunits in DNA pol III.

So, the message is that DNA polymerase is not a simple enzyme; it’s a
complicated multifunctional enzyme.

 [slide 38]: Clamping protein

A DNA clamp functions to keep DNA pol III in contact with the
template DNA strand during replication. Clamping protein is
composed of two things; something called a sliding protein and the
other is called loader clamp.

DNA replication in eukaryotics, principally, the concept is the same as in

prokaryotics but the enzymes are different, so you are going to see
different types of DNA polymerases.

By referring to [slide 39]:

1) DNA-pol α (alpha) is analogous to DNA-G enzyme in prokaryotics.

It initiates replication and synthesizes a primer.

2) DNA-pol β (Beta) is responsible for the repairing and it is

analogous to DNA pol I in prokaryotics.

3) DNA-pol ϒ (gama) is responsible for polymerization in

mitochondria.

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 4) DNA-pol δ (Delta) is the principle enzyme for elongation and the
synthesis of leading and lagging strands and it is analogous to
DNA pol III in prokaryotics.

5) DNA-pol ε (Epsilon) is analogous to DNA pol I in prokaryotics and

It is responsible for proofreading and filling the gaps.

 Question: Is it good or bad to have different enzymes from

prokaryotes?

Answer: It is good! If we want to attack infectious microorganisms, we

could attack those enzymes which are prokaryotic enzymes causing the
infection, without affecting the eukaryotic DNA replication of normal
cells.

 [Slide 40]: Primase or DNA-G is able to synthesize RNA primers.

 Question: what is a primer?

Answer: It is a segment of nucleotides sequences (RNA or protein as

we've said in previous lectures) that provide free 3´-OH groups to DNA
pol III to be able to do its work.

 [slide 43]: Helicase

Helicase or DNA-B functions in the unwinding of DNA strands

ahead to the moving replication fork. They destabilize the
interactions between complementary base pairs with the
assistance of DNA-A and DNA-C at the expense of energy.

 [slide 44]: SSB protein

SSB (Single Stranded-DNA Binding protein): It is a protein (not an

enzyme) that maintains the DNA template: stabilizes the single

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 strand during replication and prevent rewinding again. This protein
does not consume ATP and does not exhibit ant enzymatic
activities.

 [slide 45] : Topoisomerase

Topoisomerase cancels the supercoiling; it converts the positive

supercoils to negative supercoils and the negative supercoils to
relaxed forms (at the expense of energy); because once the
supercoiling is formed, DNA replication will stop.

 [slide 47] : steps of topoisomerase process with the consumption of

ATP:

1) Breakage of one or both strands of DNA.

2) Passage of a segment of DNA through this break in a way to

release the supercoil constraint.

3) Resealing of the DNA break.

 [Slides 48-49-50]: comparison between the two types of

topoisomerases.

Type Topoisomerase I Topoisomerase II

Another name ω -protein Gyrase
It cuts a phosphoester It cuts phosphoester
bond on one DNA bonds on both strands
strand rotates the of dsDNA, releases
broken DNA freely the supercoil
Mechanism
around the other constraint, and
strand to relax the reforms the
constraint, and phosphoester bonds.
reseals the cut

Topoisomerase has a clinical importance because it could be used to

stop the replication of cancer cells or infectious cells by inhibiting the

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topoisomerases of these cells, so that the positive supercoiling isn’t
released then the replication will stop.

 [slide 51]: DNA Ligase

DNA pol I cannot seal the gap between the two adjacent Okazaki
fragments after the RNA primer is removed and the gap is filled.
This job is carried out by DNA ligase, which catalyzes the
formation of a 3', 5'-phosphodiester bond in an ATP-dependent
reaction.

 [slide 53]: Replication fidelity

We have two mechanisms to ensure the replication fidelity:

• proofreading and correction by DNA pol I

• Base selection

 [Slide 57]: Stages of DNA replication

DNA replication process can be summarized by three stages:

1-Initiation

2-Elongation

3-Termination

[Slide 58]: Initiation stage

The origin of replication must be recognized by the enzyme DNA-A

then DNA-B and DNA-C will bind and start separating the ddDNA at

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 the origin of replication making the replication fork. After that SSB
protein will bind to the single strand and stabilize it, then DNA-G
or primase will synthesize the primer, after that DNA pol III will
starts the polymerization activity. By this step initiation is ended.

Elongation stage

DNA Pol III synthesizes and extends the two new strands (the
leading and lagging strands) till it reaches a signal to terminate the
process (we’ll talk about replication regulation later).

 [slide 59]: Genom of E.coli

This is a genome of E.coli. It has one origin of replication (at number

80) where DNA-A binds then to initiate the replication process.

 [slide 60]: Structure of origin of E.coli

This origin is composed of three tandom repeats and four

interspersed repeats. These sequences are consensus (the same
sequence in all prokaryotic cells, so they will be recognized by
DNA-A then initiating the process by DNA-B and DNA-C).

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[slide 61-65] doctor just showed them without explaining because they
were discussed in the previous lecture.

 [slide 66]: Elongation stage

It starts at the same time in both strands. Two monomers of DNA

pol III with its three subunits (α , ε , and θ ) will be present; one
monomer will work on the leading strand and another one on the
lagging strand at the same time in 5’----3’ direction.

 How can both strands leading (5’----3’) and lagging (3’---5’) be

synthesis in the same direction??

 [slide 68]: Mechanism of elongation of both strands in the

same direction

As you see here, the leading strand is synthesized in 5'--- 3'

direction and the looping of the lagging strand template by 180⁰
brings it to the same orientation as the leading strand template.

When the being-synthesized okazaki fragment reaches the 5' end

of the previous already-synthesized okazaki fragment, the lagging
strand template is released and unlooped, then the enzyme will
attach to another primer and loop it to be in the same orientation
of the leading strand.

 [slide 71]: Termination

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 DNA pol I works at the end of the Okazaki fragment and further
elongates the DNA chain while simultaneously removing the RNA
primer with its 5’ to 3’ exonuclease activity. However, DNA pol I
cannot seal the gap between the two adjacent Okazaki fragments.
This job is carried out by DNA ligase, which catalyzes the formation
of a 3', 5'-phosphodiester bond in an ATP-dependent reaction.

 [slide 77]: Telomeres

Telomeres are the terminal ends of chromosomes. They are

specific repetitive DNA sequences, these tandom repeats can be
several thousand nucleotides long with multiple copies of G and T
sequences. With multiple cycles of replication, the strands will
shorten until the genes, near the end on chromosome, are started
to be deleted, thus affecting the organism functions.

In order to avoid this problem (shortening of the chromosomes), there is

an enzyme called telomerase that will help to extend the telomeric
sequences by providing a movable template.

As you can see in (slide 76), after termination we'll get the leading and
the lagging strands shorter than the parental strands.

 Note: in the book and the Wikipedia, it is ensured that only the
lagging strand shortens and the leading doesn't, so check it from
the doctor to get the correct answer.

Telomerase of cancer cells are highly active, so after each replication

cycle the telomerase do its function and maintains the length of
chromosome. Consequently, cancer cells don't die by themselves. So,
one of the strategies to treat cancer is to inhibit telomerase, so that the
chromosome will be shortening and finally destroyed.

Rule of telomerase enzyme in aging:

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Telomerase with age will be less active, so with age, cells will
degenerate. This is an explanation of aging process.

Dr. said: my telomerase activity is less than your telomerase activity.

 A student asked: Why there is shortening in the leading and lagging

strands in each replication cycle in eukaryotes? What is the reason?

 [slide 76]:

Gaps result from removal of the primers that were used to start the
replication and the problem is in the fact that DNA polymerase can
only add nucleotides to the 3' end of preexisting polynucleotides
and we don't have a system that adds to 5' end.

Prokaryotics don't have this problem because they do have circular DNA
with no ends, although its DNA polymerase fails to work in the 3'- 5'
direction too.

So telomeres have only repeated sequences (no genes) to protect the

gene expression.

 Question: anti-cancerous drugs that interfere with telomerase

activity take, relatively, a long time till the telomeres shorten then
after that the genes start to be affected and lost. So it is not an
effective treatment, is it??

Answer: In cancer, cells have uncontrolled fast DNA replication and we

reach the target (genes) quickly.
Note: Telomerase activity is absent in somatic cells, while it is needed in
germ cells.

 [slide78]: Structure of telomerase

Telomerase is composed of RNA, protein and reverse

transcriptase.

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  Question: Why do we need RNA in the telomerase enzyme?

To answer this question lets see this video about TELOMER REPLICATION.

http://www.youtube.com/watch?v=AJNoTmWsE0s&p

Lastly, the doctor summarized all the stages of replication quickly and
you can find that summary in the slides.

Done By:
Hanan Al-Fayomi

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