CHAPTER10

1. CHAPTER10 Homologous Recombination at the Molecular Level 200431760044 顾辉辉 生物科学第四组

2. Outline • Models for homologous recombination • Homologous recombination protein machines • Homologous recombination in eukaryotes • Mating-type switching • Genetic consequences of the mechanism of homologous recombination

3. Models for homologous recombination Key steps of homologous recombination shared by different models • Alignment of two homologous DNA molecules • Introduction of breaks in the DNA • Formation of initial short regions of base pairing between the two recombining DNA molecules • Movement of the Holliday junction • Cleavage of the Holliday junction

4. The Holliday model illustrates key steps in homologous recombination The Holliday model very well illustrates the DNA strand invasion, branch migration, and ,Holliday junction resolution processes central to homologous recombination

5. Holliday model through the steps of branch migration

6. Holliday junction cleavage

7. Double –stranded DNA breaks arise by numerous means and initiate homologous recombination • Double-straned breaks in DNA arise quite frequently • In bacteria, the major biological role of homologous recombination is to repair DSBs • In addition to repairing DSBs in chromosomal DNA, homologous recombination promotes genetic exchange in bacteria • In eukaryotic cells, homologous recombination is critical for repairing DNA

8. Damage in the DNA template canlead To DSB formation during DNA replication

9. Homologous recombination protein machines • The recBCD helicase nuclease processes broken DNA molecules for recombination • RecA protein assembles on single-stranded DNA and promotes strand invasion • Newly base-paired partners are eatablidshed within the recA filament • RuvAB complex specifically recognizes Holliday junctions and promotes branch migration • RuvC cleaves specific DNA strands at the Holliday junction to finish recombination

10. The recBCD helicase nuclease processes broken DNA molecules for recombination • DNA molecules with single-stranded DNA extensions or tails are the preferred substrate for initiating strand exchange between regions of homologous sequence • The RecBCD enzyme processes broken DNA molecules to generate these regions of ssDNA • RecBCD provide a means for cells to choose

11. Steps of DNA processing By RecBCD

12. Polar action of chi

13. RecA protein assembles on single-stranded DNA and promotes strand invasion • RecA is the central protein in homologous recombination • RecA makes DNA pairing and strand -exchange • The active form of RecA is a protein-DNA filaments

14. Examples of DNA paring and strand -exchange reaction

15. Three view of the RecA filament

16. Polarity of RecA assembly

17. Newly base-paired partners are established within the RecA filament RecA-catalyzed strand exchange can be divided into different stages • cross-section of single DNA strand bound to RecA protein • DNA in secondary site is testes for complementarity • Base -paring between strands is switched

18. Model of two steps in search for homology and DNA strand exchange within the RecA filament

19. RecA homologous are present in all organisms Strand-exchange proteins of the RecA family are present in all forma of life

20. RecA-like protein in three branches of life

21. RuvAB complex specifically recognizes Holliday junctions and promotes branch migration • RuvA protein is a Holliday junction specific DNA-binging protein that recognizes the structure of the DNA junction • RuvB is a hexametric ATPase

22. High resolution structure of the RuvA-DNA complex and schematic model of the RuvAB complex bound to Holliday junction DNA

23. RuvC cleaves specific DNA strands at the Holliday junction to finish recombination • Completion of recombination requires that the Holliday junction between the two recombining DNA molecules be resolved • RuvC specifically nicks two of the homologous DNA strands that have the same polarity • RuvC cleaves DNA with modest sequence specificity

24. High resolution structure of the RuvC resolves and schematic model of the RuvC dimer bound to Holliday junction DNA

25. Homologous recombination in eukaryotes • Homologous recombination has additional functions in eukaryotes • Homologous recombination is required for chromosome segregation during meiosis • Programmed generation of double –stranded DNA breaks occurs during meiosis • MRX protein processes the cleaved DNA ends for assembly of the RecA-like strand –exchange proteins • Dmc1 is a RecA-like protein that specifically functions in meiotic recombination • Many protein function together to promote meiotic recombination

26. Homologous recombination has additional functions in eukaryotes • Homologous recombination can repair double –stranded breaks in DNA , to restart collapsed replication forks, and to allow a cell’s chromosomal DNA to recombine with DNA that enters via phage infection or junction • During meiosis, homologous recombination is required for proper chromosome pairing

27. Homologous recombination is required for chromosome segregation during meiosis • Recombination must be complete before the first nuclear division to allow the homologs to properly align and then separate • The improper segregation of chromosomes, called nondisjunction, leads to a large number of gametes without the correct chromosome complement • The homologous recombination events that occur during meiosis are called meiotic recombination

28. DNA dynamics during meiosis

29. Cytological view of crossing over

30. Programmed generation of double –stranded DNA breaks occurs during meiosis • The spo11protein cuts the DNA at many chromosomal locations • The mechanism of DNA cleavage • The cleavage involves a covalent protein –DNA complex has two consequences

31. Mechanism of cleavage by spo11

32. MRX protein processes the cleaved DNA ends for assembly of the RecA-like strand –exchange proteins • The DNA a5 5he site Spo11-cataluzed double-strand break is processed to generate dingle-stranded regions needed for assembly aof the RecA-like strand-exchange protein • Processing of the DNA at the break site occurs exclusively on the DNA strand that terminates with a 5’ end

33. Overview of meiotic recombination pathway

34. Dmc1 is a RecA-like protein that specifically functions in meiotic recombination • Eukaryotes encode two well-characterized homologs of the bacterial RecA protein: Rad51 and Dmc1 • Dmc1 is expressed only as cells enter meiosis • strand exchange during meiosis occurs between a particular type of homologous DNA partner

35. Dmc1-dependent recombination occurs preferentially between nonsister homologous chromatids

36. Many protein function together to promote meiotic recombination • Some large protein-DNA complexes, known as recombination factories • Rad51, Dmc1, Rad52……

37. Co –localizations of the rad51 and Dmc1 proteins to “recombination factories” in cells undergoing meiosis

38. mating-type switching • Mating-type switching is initiated by a site-specific double-strand break • Mating-type switching is a gene conversion event, not associated with crossing over

39. Mating-type switching is initiated by a site-specific double-strand break • Mating-type switching is initiated by the introduction of a DSB at the MAT locus • This reaction is performed by a specialized DNA-cleaving enzyme, called the HO endonuclease • Mating-type switching is unidirectional

40. Genetic loci encoding mating-type information

41. Mating-type switching is a gene conversion event, not associated with crossing over • Models for recombination that do not involve Holliday junction intermediates better explain mating-type switching • Synthesis-dependent strand annealing • Completing recombination requires that the other “old” DNA strand present at MAT be removed

42. Recombination model for mating-type switching: synthesis-dependent strand annealing

43. Genetic consequences of the mechanism of homologous recombination • A corollary of the fact that recombination is generally independent of sequence is that the frequency of recombination between any two genesis generally proportional to the distance between those genes • A region of DNA does not have the “average” probability of participation in recombination • Gene conversion occurs because DNA is repaired duringrecombination

44. Comparison of the genetic and physical maps of a typical region of a yeast chromosome

45. Mismatch repair of heteroduplex DNA within recombination intermediates can give rise to gene conversion