CHAPTER 5 DNA REPLICATION, REPAIR AND RECOMBINATION

1. CHAPTER 5 DNA REPLICATION, REPAIR AND RECOMBINATION • THE MAINTENANCE OF DNA SEQUENCES • DNA REPLICATION MECHANISMS • THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES • DNA REPAIR • GENERAL RECOMBINATION • SITE-SPECIFIC RECOMBINATION

2. THE MAINTENANCE OF DNA SEQUENCES • Mutation Rates Are ~1/109 bp • Inherent fidelity of DNA polymerase is ~ 1/106 bp • Other mechanisms, proofreading and repair are necessary • Low Mutation Rates Are Necessary for Life as We Know It

3. Mutation rates are relatively constant but proteins evolve at different rates • Proteins evolve different at different rates depending on structural and functional constrains • In some proteins most changes interfere with function • In others many sequence changes are tolerated

4. DNA REPLICATION MECHANISMS • Base-pairing Underlies DNA Replication and DNA Repair • DNA Replication is always in the 5’-to-3’ Direction • The DNA Replication Fork Is Asymmetrical • Fidelity of DNA Replication Requires Several Proofreading Mechanisms

5. Base-pairing Underlies DNA Replication and DNA Repair

6. DNA replication is semiconservative

7. Fidelity of DNA Replication Requires Proofreading

8. Many polymerases contain separate 3’->5’ editing domains

9. The DNA Replication Fork Is Asymmetrical • Leading and lagging strands have different requirements

10. Accesssory proteins • DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand • Helicases - Open Up the DNA Double Helix in Front of the Replication Fork • Single strand binding proteins keep ssDNA out of trouble • Clamp subunits tether A Moving DNA Polymerase to the DNA • The Proteins at a Replication Fork Cooperate to Form a Replication Machine

11. DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand

12. Helicases - Open Up the DNA Double Helix

13. Single strand binding proteins keep ssDNA out of trouble

14. Clamp subunits tether A Moving DNA Polymerase to the DNA

15. Clamp loading and unloading on the lagging strand

16. The replication machine - expanded

17. The replication machine - trombone model

18. The eucaryotic fork is like the procaryotic but with more specialization

19. Other factors operate away from the replication fork • A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Machine • DNA Topoisomerases Prevent DNA Tangling During Replication • All topoisomerases form transient covalent phospho-tyrosine bonds to DNA backbone • Type 1 topoisomerase nicks only one strand - unwinds only • Type 2 topoisomerase nicks both strands - unwinds and untangles - Makes DNA “ethereal”

20. A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Fork

21. DNA Topoisomerase Prevents DNA Tangling During Replication

22. Type 1 topoisomerase nicks only one strand - unwinds only

23. Type 2 topoisomerase nicks both strands - Unwinds and Untangles - Makes DNA “ethereal”

24. THE INITIATION OF DNA REPLICATION • DNA Synthesis Begins at Replication Origins • BacteriaHave a Single Origin • Eucaryotic Chromosomes Contain Multiple Origins • In Eucaryotes DNA Replication Takes Place During Only One Part of the Cell Cycle • Different Regions on the Same Chromosome Replicate at Distinct Times in S Phase • Highly Condensed Chromatin Replicates Late,While Genes in Less Condensed Chromatin Tend to Replicate Early

25. DNA Synthesis Begins at Replication Origins

26. BacteriaHave a Single Origin

27. Initiation and mismatch repair in bacteria are both controlled by methylation

28. Origins can be mapped by pulse labeling

29. Different DNA regions have distinct timings of replication during the S phase of the eukaryotic cell cycle

30. Well-defined DNA Sequences Serve as Replication Origins in a Simple Eucaryote, the Budding Yeast • A Large Multisubunit Complex Binds to Eucaryotic Origins of Replication • The Mammalian DNA Sequences That Specify the Initiation of Replication Have Been Difficult to Identify • New Nucleosomes Are Assembled Behind the Replication Fork

31. Yeast Origins have been identified by genetic means

32. A Large Multisubunit Complex Binds to Eucaryotic Origins of Replication

33. Chromatin is assembled following replication forks

34. COMPLETION OF DNA REPLICATION IN CHROMOSOMES • Telomerase Replicates the Ends of Chromosomes • Telomere Length Is Regulated by Cells and Organisms

35. Telomerase is a Reverse transcriptase that carries its own RNA template

36. Telomerase solves the problem of incomplete lagging strand synthesis

37. Telomeres are sequestered in special chromatin structures

38. Many Somatic Cells have low telomerase atctivity, Some cancer cells have enhanced acivity

39. DNA REPAIR • Without DNA Repair, Spontaneous DNA Damage Would Rapidly Change DNA Sequences • The DNA Double Helix Is Readily Repaired • DNA Damage Can Be Removed by More Than One Pathway • The Chemistry of the DNA Bases Facilitates Damage Detection • Double-Strand Breaks are Efficiently Repaired • Cells Can Produce DNA Repair Enzymes in Response to DNA Damage • DNA Damage Delays Progression of the Cell Cycle

40. Nucleotides in DNA are susceptible to many types of damage

41. Depurination and Cytosine Deamination

42. Replication “fixes” mutations on one strand

43. Thymine dimers can be formed after UV irradiation

44. Excision repair

45. Double-strand breaks are the most serious lesions

46. GENERAL RECOMBINATION • General Recombination Is Guided by Base-pairing Interactions Between Two Homologous DNA Molecules • Meiotic Recombination Is Initiated by Double-strand DNA Breaks • DNA Hybridization Reactions Provide a Simple Model for the Basepairing Step in General Recombination • The RecA Protein and its Homologs Enable a DNA Single Strand to Pair with a Homologous Region of DNA Double Helix • There Are Multiple Homologs of the RecA Protein in Eucaryotes, Each Specialized for a Specific Function • General Recombination Often Involves a Holliday Junction • General Recombination Can Cause Gene Conversion • General Recombination Events Have Different Preferred Outcomes in Mitotic and Meiotic Cells • Mismatch Proofreading Prevents Promiscuous Recombination Between Two Poorly Matched DNA Sequences

47. Homologous recombination repairs spontaneous breaks in DNA and those induced in Meiotic crossing-over

48. Synapsis:Alignment of complementary DNA strands by base pairing

49. Structure and Mechanism of the RecA protein from E. coli

50. Robin Holliday’s Model for a double strand crossover junction