1. Homologous Recombination at the Molecular Level
CHAPTER10
Homologous Recombination at the Molecular Level
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 can lead 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