1. Principles of Biochemistry Fourth Edition Chapter 20 DNA Replication, Repair, and Recombination Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry Rawn

3. There are three distinct stages in DNA replication. (1)Initiation begins with the correct assembly of the replication proteins at the site where DNA replication is to start. (2) During the elongation stage, DNA is replicated semiconservatively as the complex catalyzes the incorporation of nucleotides into the growing DNA strands. (3) Finally, when replication terminates, the protein machine is disassembled, and the daughter molecules separate so that they can segregate into their new cells.

4. Bidirectional DNA replication in Escherichia coli.

5. Autoradiograph of a replicating E. coli chromosome.

6. Electron micrograph of replicating DNA from an embryo of the fruit fly Drosophila melanogaster.

8. Diagram of the subunit composition of E. coli DNA polymerase III.

9. Elongation of a DNA chain.

11. Model of bacteriophage DNA polymerase bound to DNA.

12. Diagram of the replication fork.

13. Discontinuous DNA synthesis demonstrated by analysis of newly synthesized DNA.

14. Diagram of lagging-strand synthesis.

15. Structure of the Klenow fragment with a bound DNA fragment.

16. Joining of Okazaki fragments by the combined action of DNA polymerase I and DNA ligase.

18. Nick translation

19. Joining of Okazaki fragments by the combined action of DNA polymerase I and DNA ligase.

20. Proposed mechanism of DNA ligase in E. coli Nicotinamide mononucleotide (NMN+)

22. Simultaneous synthesis of leading and lagging strands at a replication fork. Replisome: DNA polymerase III holoenzyme (only the core complexes are shown) single-strand binding protein helix-destabilizing protein primase, a helicase, and other subunits

23. Simultaneous synthesis of leading and lagging strands at a replication fork. SSB tetramer covers 32 nucleotides of DNA. Binding of SSB to DNA is cooperative; that is, binding of the first tetramer facilitates binding of the second, and so on.

24. Simultaneous synthesis of leading and lagging strands at a replication fork.

26. Location of the origin (oriC) and terminus (ter) of DNA replication in E. coli. dnaA is the gene for the protein DnaA, which is required to initiate replication. The distance between oriC and dnaA is about 40 kb. The red arrows indicate the direction of movement of the replication forks.

27. Structure of E. coli Tus bound to DNA. Tus: terminator utilization substance Tus binds to specific sequences at the termination site of DNA replication. The bound protein blocks movement of the replisome.

29. This cell cycle is a highly regulated progression through a series of dependent steps that at a minimum accomplishes two goals: (1) it faithfully duplicates all of the DNA in a cell to produce exactly two copies of each chromosome, (2) it precisely segregates one copy of each replicated chromosome into one of the two daughter cells.

30. The eukaryotic cell division cycle coordinates DNA replication and mitosis S phase: DNA replicated, synthesized M phase: cell mitosis G1: each ORC stimulates the formation of a pre-replication complex (pre-RC, involves Cdc6, Cdt1 and helicase MCM). Until the activity of an S-phase protein kinase (SPK) reaches a critical threshold, whereby the initiation complex recruits waiting replisomes and the origin is said to “fire”.

31. Sanger method for sequencing DNA.

32. Repair of Damaged DNA DNA is the only cellular macromolecule that can be repaired. Why????

33. A defective protein forms as a result of a translation error; simply replaced by a new, functional protein. When DNA is damaged In single-celled organisms, damage to a gene encoding an essential protein may kill the organism. In multicellular organisms, the accumulation of defects in DNA over time can lead to progressive loss of cellular functions or to deregulated growth such as that seen in cancer cells.

34. Photodimerization of adjacent deoxythymidylate residues. Ultraviolet light causes the bases to dimerize, thus distorting the structure of DNA thymine dimer

35. Repair of thymine dimers by DNA photolyase – direct repair.

36. General excision-repair pathway. Other forms of ionizing radiation and naturally occurring chemicals can damage DNA. Some compounds, including acids and oxidizing agents, can modify DNA by alkylation, methylation, or deamination. DNA is also susceptible to spontaneous loss of heterocyclic bases, a process known as depurination or depyrimidization. General excision-repair pathway. UvrABC 12-13 nt UvrABC can also repair the pyrimidine dimer.

37. Hydrolytic deamination of cytosine Spontaneous deamination of cytosine is one of the most common types of DNA damage. Deamination of cytosine produces uracil, which pairs with adenine rather than guanine.

38. Uracil N-glycosylase from human mitochondria The enzyme is bound to a uracil-containing nucleotide (green) that has been flipped out of the stacked region of double-stranded DNA. DNA glycosylases remove deaminated bases and some other modified bases by catalyzing hydrolysis of the N- glycosidic bonds that link the modified bases to the sugars.

39. Repair of damage resulting from the deamination of cytosine. apurinic and apyrimidinic sites

40. Repair of damage resulting from the deamination of cytosine. AP-endonuclease

41. Repair of damage resulting from the deamination of cytosine.

42. Homologous Recombination None of your children’s chromosomes, for example, will be the same as yours, and none of yours are the same as those of your parents’.

43. Holliday model of general recombination. First, nicks are introduced into a homologous region of each molecule. Subsequent strand invasion, DNA cleavage at the crossover junction, and sealing of nicked strands result in exchange of the ends of the chromosomes Ruv C

44. Recombination in E. coli 1. RecBCD endonuclease generats the single-stranded DNA with a free end. It then unwinds the DNA in a process coupled to ATP hydrolysis, generating single-stranded DNA with a 3’ terminus. 2. RecA binds the single-stranded DNA and promotes the formation of triple strand intermediate. 3. Strand invasion and displacement occur.

45. Strand exchange catalyzed by RecA.

46. Action of Ruv proteins at Holliday junctions.

47. Model of RuvA and RuvB bound to a Holliday junction.

48. Branch migration and resolution.

49. Recombination Can Be a Form of Repair RecA-mediated strand exchange between the homologous daughter chromosomes allows the intact strand from one daughter molecule to act as a template for repairing the broken strand of the other daughter molecule. Recombination also creates new combinations of genes on a chromosome, an added bonus for the population and its chances for evolutionary survival.