1. DNA Replication Lecture 10 Dr. Attya Bhatti

2. Mechanism of DNA replication In E-Coli In the late 1950s, Arthur Kornberg successfully identified and purified the first DNA polymerase, an enzyme that catalyzes the replication reaction. The total amount of DNA at the end of the reaction can be as much as 20 times the amount of original input DNA, so most of the DNA present at the end must be progeny DNA. DNA polymerases:

3. There are three DNA polymerases in E. coli. The first enzyme that Kornberg purified is called DNA polymerase I or pol I. This enzyme has three activities...... 1. a polymerase activity, which catalyzes chain growth in the 5′ → 3′ direction; 2. a 3′ → 5′ exonuclease activity, which removes mismatched bases; 3. A 5′ → 3′ exonuclease activity, which degrades double-stranded DNA. Mechanism of DNA replication In E-Coli

4. Pol II may repair damaged DNA, although no particular role has been assigned to this enzyme. Pol III, together with pol I, has a role in the replication of E. coli DNA. Mechanism of DNA replication In E-Coli Figure: DNA replication fork.

5. Prokaryotic origins of replication: E. coli replication begins from a fixed origin but then proceeds bidirectionally ending at a site called the terminus. The unique origin is termed oriC. It is 245 bp long and has several components. Mechanism of DNA replication In E-Coli

6. Figure: Chain-elongation reaction catalyzed by DNA polymerase.

7. First, there is a side-by-side, or tandem, set of 13-bp sequences, which are nearly identical. There is also a set of binding sites for a protein, the DnaA protein. An initial step in DNA synthesis is the unwinding of the DNA at the origin in response to binding of the DnaA protein. Mechanism of DNA replication In E-Coli

8. Figure: OriC, the origin of replication in E. coli, has a length of 245 bp. It contains a tandem array of three nearly identical 13-nucleotide sequences and four binding sites for DNA protein.

9. Figure: Bidirectional replication of a circular DNA molecule.

10. Replication-division cycle in Bacteria is 25-30-minute Eukaryotes can vary from 1.4 hours in yeast to 24 hours in cultured animal cells and may last from 100 to 200 hours in some cells. Eukaryotes have to solve the problem of coordinating the replication of more than one chromosome, as well as replicating the complex structure of the chromosome itself. Eukaryotic origins of replication

11. In eukaryotes, replication proceeds from multiple points of origin. Replication appears to begin at several different sites on eukaryotic chromosomes. Eukaryotic origins of replication Figure: Replication pattern in a Drosophila polytene chromosome revealed by autoradiography. Several points of replication are seen within a single chromosome, as indicated by arrows.

12. Yet there is no firm proof that these regions are indeed different starting points on a single DNA molecule. However, experiments in yeast indicate the existence of approximately 400 replication origins distributed among the 17 yeast chromosomes, and in humans there are estimated to be more than 10,000 growing forks. Eukaryotic origins of replication

13. Priming DNA polymerases can extend a chain but cannot start a chain. Therefore, as already mentioned, DNA synthesis must first be initiated with a primer, or short oligonucleotide, that generates a segment of duplex DNA. RNA primers are synthesized either by RNA polymerase or by an enzyme termed primase. Primase synthesizes a short (approximately 30 bp long) stretch of RNA complementary to a specific region of the chromosome.

14. Figure: Initiation of DNA synthesis by an RNA primer.

15. The RNA chain is then extended with DNA by DNA polymerase. E. coli primase forms a complex with the template DNA, and additional proteins, such as DnaB, DnaT, Pri A, Pri B, and Pri C. The entire complex is termed a primosome.Priming

16. Leading strand and lagging strand DNA polymerases synthesize new chains only in the 5′ → 3′ direction. So one new strand is synthesized continuously called leading strand. The other synthesized in short, discontinuous segments, called lagging strand. Thus, the new strand must grow in a direction opposite that of the movement of the replication fork.

17. Figure: DNA synthesis proceeds by continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.

18. In E. coli, pol III carries out most of the DNA synthesis on both strands, and pol I fills in the gaps left in the lagging strand, which are then sealed by the enzyme DNA ligase. DNA ligases join broken pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5′ phosphate end of a hydrogen- bonded nucleotide and an adjacent 3′ OH group. The RNA primer is removed at a later stage of replication. Leading strand and lagging strand

19. Figure: Coordination between the Leading and the Lagging Strands. The looping of the template for the lagging strand enables a dimeric DNA polymerase III holoenzyme to synthesize both daughter strands. The leading strand is shown in red, the lagging strand in blue, and the RNA primers in green. One proposed mechanism that allows the same dimeric holoenzyme molecule to participate in both leading- and lagging-strand synthesis is the looping of the template for the lagging strand allows a single pol III dimer to generate both daughter strands. After approximately 1000 base pairs, pol III will release the segment of lagging-strand duplex and allow a new loop to be formed.

20. Figure: Schematic representation of the enzymatic events at a replication fork in E. coli. Enzymes shaded in yellow catalyze chain initiation, elongation, and ligation. The wavy lines on the lagging strand denote RNA primers.

21. The primers for the discontinuous synthesis on the lagging strand are synthesized by primase (step a). The primers are extended by DNA polymerase (step b) to yield DNA fragments that were first detected by Reiji Okazaki and are termed Okazaki fragments. The 5′ → 3′ exonuclease activity of pol I removes the primers (step c). And fills in the gaps with DNA, which are sealed by DNA ligase (step d). Leading strand and lagging strand

22. Replication at chromosome tips Figure: The replication problem at chromosome ends. There is no way of priming the last section of the lagging strand, and a shortened chromosome would result.

23. The ends of chromosomes present a special problem for the replication process. for the leading strand, the polynucleotide addition during replication can always extend to the end because it is automatically primed from behind. However, at the tip, the lagging strand reaches a point where its system of RNA priming cannot work, and an unpolymerized section remains and a shortened chromosome would be the result. Replication at chromosome tips

24. To solve this problem, the tips of chromosomes, have adjacent repeats of simple DNA sequences, called telomeres. E.g. In humans it is TTAGGG. These repeats do not code for an RNA or a protein product but nevertheless serve a definite function in replication. An enzyme called telomerase adds these simple repeat units to the chromosome ends. Replication at chromosome tips

25. Figure. Telomerase carries a short RNA molecule that acts as a template for the addition of the complementary DNA sequence at the 3′ end of the double helix.

26. An age-dependent decline in telomere length has been found in several somatic tissues in humans. In addition, human fibroblasts in culture show progressive telo-mere shortening up to their eventual death. Such observations have led to the telomere theory of aging, and the validity of this theory is now being tested. Replication at chromosome tips

27. Helicases and topoisomerases Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA strands together in a double helix. Among E. coli helicases are the DnaB protein and the Rep protein. The unwound DNA is stabilized by the single- stranded binding (SSB) protein, which binds to the single-stranded DNA and retards reformation of the duplex.

28. The action of helicases during DNA replication generates twists in the circular DNA that need to be removed to allow replication to continue. Circular DNA can be twisted and coiled, much like the extra coils that can be introduced into a rubber band. This supercoiling can be created or relaxed by enzymes termed topoisomerases. E.g. DNA Gyrase Helicases and topoisomerases

29. Eukaryotic DNA polymerases There are at least five DNA polymerases, α, β, γ, δ, and ε, in higher eukaryotes. Polymerases α and δ in the nucleus have roles similar to pol I in E. coli. Polymerase β has a role in DNA repair and gap filling. The γ polymerase is found in mitochondria and appears to take part in replication of mitochondrial DNA.

30. Problem Q. If the GC content of a DNA molecule is 56 percent, what are the percentages of the four bases (A, T, G, and C) in this molecule?