CHAPTER 25 DNA Metabolism

1. CHAPTER 25DNA Metabolism DNA replication DNA repair DNA recombination Key topics:

2. What is DNA Metabolism? While functioning as a stable storage of genetic information, the structure of DNA is far from static: A new copy of DNA is synthesized with high fidelity before each cell division Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made Segments of DNA are rearranged either within a chromosome or between two DNA molecules giving offspring a novel DNA DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

3. DNA Metabolism • DNA replication: processes by which copies of DNA molecules are faithfully made. • DNA repair: processes by which the integrity of DNA are maintained. • DNA recombination: processes by which the DNA sequences are rearranged.

4. Map of the E. coli chromosome.

5. DNA Replication Is Semiconservative.

6. Replication Forks may Move Either Unidirectionally or Bidirectionally

8. Replication Begins at an Origin and Proceeds Bidirectionally in Many Bacteria Such as E. coli.

9. DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cathion such as Mg++.

10. DNA Elongation Chemistry Parental DNA strand serves as a template Nucleotide triphosphates serve as substrates in strand synthesis Hydroxyl at the 3’ end of growing chain makes a bond to the -phosphorus of nucleotide Pyrophosphate is a good leaving group

12. DNA Synthesis Can’t be Continuously on Both Strands (because the DNA duplex is antiparallel and all DNA polymerases synthesize DNA in a 5’ to 3’ direction) What is the source of primer used for lagging strand synthesis?

13. DNA Replication is Very Accurate • Base selection by DNA polymerase is fairly accurate (about 1 error per 104) • Proofreading by the 3’ to 5’ exonuclease associated with DNA polymerase improves the accuracy about 102 to 103-fold. • Mismatch repair system repairs any mismatched base pairs remaining after replication and further improves the accuracy.

14. An Example of Proofreading by the 3’ to 5’ Exonuclease of DNA Polymerase I of E. coli

16. Large (Klenow) fragment of DNA polymerase I retains polymerization and proofreading (3’ to 5’ exo)

17. DNA polymerase I has 5’ to 3’ exonuclease and can conduct Nick Translation

18. PolIII*consists of two cores, a clamp-loading complex (g complex) consisting of t2gdd’, and two additional proteins c and y. Holoenzyme is PolIII* plus b subunits.

19. DNA polymerase III

20. The two b subunits of PolIII form a circular clamp that surrounds DNA

21. DNA Replication requires many enzymes and protein factors • Helicases: separation of DNA duplex. • Topoisomerase: relieves topological stress • Single-strand DNA binding proteins: stabilizes separated DNA strands. • Primase: synthesizes RNA primer. • DNA Pol I: removes RNA in Okazaki fragments and fills the gaps between Okazaki fragments. • Ligase: seals nicks.

22. Replication of the E. coli chromosome • Initiation. • Elongation. • Termination.

23. Initiation begins at a fixed origin, called oriC, which consists of 245 bp bearing DNA sequences that are highly conserved among bacterial replication origins.

25. Model for initiation of replication at oriC.

26. Proteins involved in Elongation of DNA

27. Elongation:Synthesis of Okazaki fragments

28. Model for the synthesis of DNA on the leading and lagging strands by the asymmetric dimer of PolIII

31. Pol I can remove RNA primer and synthesize DNA to fill the gap

33. Termination: When the two opposing forks meet in a circular chromosome. Replication of the DNA separating the opposing forks generated catenanes, or interlinked circles.

34. Termination sequences and Tus (termination utilization substance) can arrest a replication fork

35. Replication in eukaryotic cells is more complex • Contains many replicons. • How is DNA replication initiated in each replicon is not well understood. Yeast cells appears to employ ARS (autonomously replicating sequences) and ORC (origin recognition complex) to initiate replication. • More than one DNA polymerase are used to replicate DNA. • End-replication problem of linear DNA.

36. Assembly of a pre-replicative complex at a eukaryotic replication origin

37. The End Replication Problem of Linear DNA

38. DNA Damages • DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism. • DNA damage may be classified as: (i) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links.

39. DNA Repair and Mutations Chemical reactions and some physical processes constantly damage genomic DNA At the molecular level, damage usually involves changes in the structure of one of the strands Vast majority are corrected by repair systems using the other strand as a template Some base changes escape repair and the incorrect base serves as a template in replication The daughter DNA carries a changed sequence in both strands; the DNA has been mutated Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens

40. Ames test for mutagens (carcinogens)

42. Methylataion and Mismatch Repair

43. Model for Mismatch Repair

45. Base-Excision Repair

46. Nucleotide-Excision Repair in E. coli and Humans

47. Direct Repair: Photoreactivation by photolyase

48. Alkylation of DNA by alkylating agents

49. O6-methyl G, if not repaired, may produce a mutation

50. Direct Repair:Reversal of O6 methyl G to G by methyltransferase