1

720

Enzymology of DNA replicationNabeel Hanan Al-Fayomi13/10/

2010

Molecular Genetics – Lecture 7Wednesday, October 13, 2010Done 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.

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

2

[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:

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

3

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.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

4

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 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 IIAnother name w-protein Gyrase

Mechanism

It cuts a phosphoester bond on one DNA strand rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut

It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.

5

Topoisomerase has a clinical importance because it could be used to stop the replication of cancer cells or infectious cells by inhibiting the 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 replicationDNA replication process can be summarized by three stages:

1- Initiation2- Elongation3- 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 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.

6

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).

[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 (a, e, and q) 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??

7

[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

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.

8

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:

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

9

Telomerase is composed of RNA, protein and reverse transcriptase.

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

10