Objectives of DNA recombination

1. Objectives of DNA recombination • The different processes of DNA recombination: homologous recombination, site-specific recombination, transposition, illegitimate recombination, etc. • What are the differences between these process: (i) the DNA substrates, (ii) the enzymes used, and (iii) the recombinant products produced. • General mechanism of recombination: (I) presynapsis (initiation), (ii) synapsis (the formation of joint molecules), and (iii) postsynapsis (resolution). • In addition to provide genetic diversity, DNA recombination plays an important role in repair of DNA double-strand breaks and DSG (to be discussed in the section of DNA repair).

2. Examples of recombination

3. Homologous recombination • Refer to recombination between homologous DNA sequence in the same or different DNA molecules. • The enzymes involved in this process can catalyze recombination between any pair of homologous sequences, as long as the size of homologous sequence is longer than 45 nt or longer. No particular sequence is required. • Models of homologous recombination. • Homologous recombination of E. coli. • Meiotic recombination.

4. The Holliday model of recombination

6. Homologous recombination of E. coli • Identification of genes involved in recombination: (i) isolation of mutants affecting recombination in wild-type cells (eg., recA, recB, recC etc.), (ii) the recombinational deficiency in recBC cells may be suppressed by sbcA or sbcB mutations. The sbcB gene encodes for a 3’ to 5’ ss-DNA exonuclease, while the sbcA mutation activate the expression of recE which encodes for 5’ to 3’ exonuclease. (iii) isolation of mutants affecting recombination in recB recC sbcB or recB recC sbcA cells (eg., recF, recO, recR, recQ, recJ etc.) • The biochemical functions of rec genes.

7. Homologous recombination is catalyzed by enzymes • The most well characterized recombination enzymes are derived from studies with E. coli cells. • Presynapsis: helicase and/or nuclease to generate single-strand DNA with 3’-OH end (RecBCD) which may be coated by RecA and Ssb. • Synapsis: joint molecule formation to generate Holliday juncture (RecA). • Postsynapsis: branch migration and resolution of Holliday juncture (RuvABC).

8. RecBCD • A multifunctional protein that consists of three polypeptides RecB (133 kDa), RecC (129 kDa) and RecD (67 kDa). • Contain nuclease (exonuclease and Chi-specific endonuclease) and helicase activity.

9. Chi-specific nicking by RecBCD 5‘-GCTGGTGG-3’ Fig. 22.7

10. Helicase and nuclease activities of the RecBCD

11. The Bacterial RecBCD System Is Stimulated by chi Sequences FIGURE 15.17: RecBCD unwinding and cleavage

12. The RecBCD pathway of recombination

13. RecA binds selectively to single-stranded DNA Fig. 22.4

14. RecA forms nucleoprotein filament on single-strand DNA

15. Fig. 22.5

16. Paranemic joining of two DNA (in contrast to plectonemic) Fig. 22.6

17. Strand-Transfer Proteins Catalyze Single-Strand Assimilation FIGURE 15.18: RecA strand invasion • RecA forms filaments with single-stranded DNA and catalyzes the assimilation of single-stranded DNA to displace its counterpart in a DNA duplex.

18. RuvABC • RuvA (22 kDa) binds a Holliday junction with high affinity, and together with RuvB (37 kDa) promotes ATP-dependent branch migration of the junctions leading to the formation of heteroduplex DNA. • RuvC (19 kDa) resolves Holliday juncture into recombinant products.

19. Fig. 22.9

20. Fig. 22.10

23. Fig. 22.14

24. Fig. 22.15

25. Fig. 22.17

28. Homologous Recombination Occurs between Synapsed Chromosomes in Meiosis FIGURE 03: Recombination occurs at specific stages of meiosis Chromosomes must synapse (pair) in order for chiasmata to form where crossing-over occurs. The stages of meiosis can be correlated with the molecular events at the DNA level.

29. Fig. 15.13

31. Fig. 15.15

32. The Synaptonemal Complex Forms after Double-Strand Breaks Double-strand breaks that initiate recombination occur before the synaptonemal complex forms. If recombination is blocked, the synaptonemal complex cannot form. Meiotic recombination involves two phases: one that results in gene conversion without crossover, and one that results in crossover products.

33. Fig. 22.18

34. Fig. 22.19

35. Fig. 22.20

36. Fig. 22.21

38. Fig. 22.24

39. Gene conversion: the phenomenon that abnormal ratios of a pair of parental alleles is detected in the meiotic products.

40. Fig. 22.25

41. Fig. 22.26

42. Site-specific Recombination: Bacteriophage lambda integration in E. coli

43. Fig. 15.28

44. A site-specific recombination reaction (eg. catalyzed by Int of bacteriophage lambda)

45. Fig. 15.31

50. Recombination Pathways Adapted for Experimental Systems FIGURE 15.38: Cre/lox system for gene knockouts Adapted from H. Lodish, et al. Molecular Cell Biology, Fifth edition. W. H. Freeman & Company, 2003.