Chapter 15 DNA Biosynthesis and DNA Damage Repair

1. Chapter 15 DNA Biosynthesis and DNA Damage Repair

2. Section One The General Features of Replication of Chromosomal DNA

3. DNA replicates semiconservatively. ⑴. Watson and Crick predicted that DNA might semiconservatively replicate.

4. the hypothesis ___________ p ___________ d ___________ p ___________ p ____________ d ____________ p

5. ⑵. In 1958 Meselson and Stahl demons- trated the semiconservative nature of DNA replication in E coli.

6. The experiment: CsCl equilibrium gradient density ultracentri- fugation of 15N labeled E coli DNA.

7. The density of DNA was increased by labeling it with 15N, a heavy isotope of nitrogen. This was done by growing E coli 15 ge- nerations in a medium that contained 15NH4Cl as its only nitrogen source.

8. 15N DNA was extracted and subjected to CsCl equilibrium gradient density ultracen- trifugation. The DNA band position was recorded. There was one band of 15N DNA.

9. The bacteria were transferred to an 14NH4Cl medium and grown for one generation. The DNA density was determined again. The position of the DNA band was compared with that of the 15N DNA .

10. What was the result ?

11. There was one DNA band. It had a lower density than 15N DNA because its position was above on that of 15N DNA. It was 15N/14N hybrid DNA.

12. After another generation growing in the 14NH4Cl medium the bacterial DNA density was determined. There were two DNA bands.

13. One half of the DNA was 14N DNA, and another half was hybrid DNA. In succeeding generations the ratio of 14N DNA to hybrid DNA increased gradually. The hybrid DNA became less and less..

14. summary DNA replicates in a semiconservative man- ner. When the two parental strands sepa- rate, each serves as the template for making a new , complementary strand.

15. 2. The point at which separation of the strands and synthesis of new DNA takes place is known as the replication fork. The replication fork is Y-shaped. Two arms (V) are separated strands which act as the template and DNA synthesis is actively taking place. The body (I) is the parental DNA.

16. 3.DNA replication is usually bidirectional. ⑴.Replicon: Any piece which replicates as a single unit is called a replicon. All bacterial chromosomes and many phage and virus DNA molecules are circular and comprise single replicons.

17. In contrast eukaryotic chromosomes consist of multiple replicons. ⑵. Origin: The initiation within a replicon always occurs at a fixed point known as the origin.

18. ⑶.Terminus: In a circular replicon there is a single termination site roughly 180° opposite the unique origin.

19. Summary In a circular replicon replication begins from the fixed origin and forms two repli-cation forks. The two replication forks proceed bidirec- tionally away from the origin and the strands are copied as they separate until the terminus is reached.

20. 4. DNA replication is semidiscontinuous. ⑴.The mechanism of DNA replication allows only for synthesis in a 5’3’ direction. ⑵. The two strands of DNA are antiparallel.

21. Question How is the parental strand that runs 5’3’ past the replication fork copied ? The answer is semidiscontinuous replica- tion.

22. At each replication fork one strand ( the lead- ing strand ), whose template runs 3’5’ past the replication fork, is synthesized as one con- tinuous piece, while the other strand ( the lag- ging strand ), whose template runs 5’3’ past the replication fork is made discontinuously as short fragments in the reverse direction.

23. These short fragments are called Okazaki fragments. They are joined by DNA ligase and form the lagging strand.

24. 5. Origins contain short AT-rich repeat se- quences. ⑴. Prokaryotic and eukaryotic origins have common features: a. They consist of multiple unique short repeat sequences. b. These sequences are recognition and binding sites of multi-subunit initiation factors.

25. c. These sequences are usually AT- rich. ⑵. E coli ‘s origin is called oriC. It is 254bp long and contains three 13-bp direct repeats and four 9-bp inverted re- peats.

26. 6. DNA replication needs priming. ⑴. DNA polymerases cannot initiate DNA replication by starting a new DNA chain. They can only add nucleotides to the 3’ end of an existent piece of RNA or DNA under the direction of the template. The existent piece of RNA or DNA are called primer.

27. ⑵. The leading strand and all Okazaki frag- ments are primed by synthesis of a short piece of RNA ( an RNA primer ), which is then elongated with DNA by DNA poly- merase. ⑶. There are also DNA priming or nucleotide priming.

28. 7.Multi-enzymes and proteins participate in DNA replication. ⑴. Topoisomerases regulate the type and level of supercoiling of dsDNA. ⑵. Helicases unwind the dsDNA. ⑶. SSBs bind and stabilize the single DNA strand. ⑷. Primase synthesizes the RNA primer.

29. ⑸. DNA polymerases elongate DNA chains. ⑹. DNA ligase joins Okazaki fragments.

30. 8. DNA replication is of high fidelity. ⑴. There are two types of replication errors. a. base ( nucleotide ) substitution. b. nucleotide insertion or deletion. ⑵. There are two types of error controls a. presynthetic error control. b. proofreading control ⑶. mismatch repair.

31. Section Two Features of DNA Polymerases

32. The substrates of DNA polymerases are

33. 2. The active center of DNA pols catalyzes DNA synthesis. ⑴. The active center can differentiate dNTP from NTP. ⑵. DNA pols can choose the right nucleotide for base-pairing with the template nucleo- tide.

34. 3. The semi-closed right-handed structure of the DNA pol. is composed of three domains. ⑴. thumb domain, fingers domain and palm domain ⑵. two active centers: polymerase active center and 3’-5’exonuclease active center. They are located in the palm domain.

35. ⑶.The palm domain has three functions: a. polymerase activity b. to check the newly formed base pair c. proofreading control to remove the ‘ mis-paired ‘ nucleotide. ⑷. The fingers domain binds the template strand and interacts with the nucleotide that enters the polymerase active center.

36. ⑸.The thumb domain keeps the primer-template junction in position in the active center and makes the polymerase bind the substrates tightly.

37. 4. The protein of ‘ sliding clamp’, that encircles the DNA and interacts with the DNA pol, is responsible for the processivity of the DNA polymerase. Processivity of the DNA pol means DNA pol going on synthesis of DNA rapidly without stopping.

38. Section Three DNA Replication in E coli

39. 1.E coli DNA replication initiates at oriC in a process mediated by a multi-protein com- plex. ⑴. Protein factors participate in initiation at oriC include: DnaA, DnaB, DnaC, HU, to- poisomerase II ( gyrase ) and SSB.

40. ⑵. DnaA protein forms a complex of 20-40 molecules, each bound to an ATP mole- cule, around which the oriC DNA with four 9-bp repeats becomes wrapped. ⑶. This facilitates melting of three 13-bp repeats which open to allow binding of DnaB protein.

41. ⑷. With the help of DnaC, DnaB binds the opened DNA. DnaB is a helicase and can unwind dsDNA by using the energy of ATP hydrolysis. ⑸. SSB binds the single DNA strand. ⑹. Gyrase introduces negative supercoils into the dsDNA ahead of the replication fork. ⑺. The prepriming complex is formed.

42. 2. Primase synthesizes RNA primer. ⑴. DnaG is a primase. It binds the template and is activated by DnaB. ⑵. The activated primase synthesizes RNA primer.

43. 3. DNA pol III elongates DNA and DNA pol I removes the primer. ⑴. DNA pol III is the principal enzyme in elongation of DNA. ⑵. The structure of the holoenzyme is composed of 10 different subunits in total number of 16. (αεθ)2ζ2γ2δδ’χψβ2

44. Two core enzymes (αεθ)2 are held together by a γcomplex. αsubunit : DNA synthesis εsubunit : proofreading βsubunit : the ‘ sliding clamp ’ A single holoenzyme is responsible for the synthesis of both leading strand and Okazaki fragments of lagging strand.

45. The holoenzyme of DNA pol III has two core enzymes. One is responsible for the synthesis of leading strand. The other for the synthesis of Okazaki fragments. Because the template of the lagging strand is looped out both leading and lagging strand synthesis move in the same direction.

46. When the lagging strand core enzyme completes an Okazaki fragment, it re- leases the strand. Then the primosome ( DnaB-DnaG com- plex ) synthesizes another primer and the core enzyme elongates and completes another Okazaki fragment.

47. ⑶. DNA pol I has only one polypeptide. It has three enzyme activities: a. the polymerase activity b. the 3’ 5’ exonuclease activity c. the 5’ 3’ exonuclease activity. Subtilicin can cut it into two fragments.

48. The large fragment is called klenow fragment. It has the polymerase activity and the 3’5’ exonuclease activity. The 5’3’ exonuclease removes the primer.

49. The polymerase function simultaneously fills the gap with DNA by elongating the 3’-end of the adjacent Okazaki fragment. The final phosphodiester bond between the fragments is made by DNA ligase.

50. 4.Tus protein recognizes and binds to the TER site. That prevents the replication fork advancing. DNA replication terminates.