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
RecA forms filaments with single-stranded DNA and catalyzes the assimilation of single-stranded DNA to displace its counterpart in a DNA duplex.
FIGURE 15.18: RecA strand invasion

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

23. Fig

24. Fig

25. Fig

28. FIGURE 03: Recombination occurs at specific stages of meiosis
Homologous Recombination Occurs between Synapsed Chromosomes in 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.
FIGURE 03: Recombination occurs at specific stages of meiosis

29. Fig

31. Fig

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

34. Fig

35. Fig

36. Fig

38. Fig

39. Gene conversion: the phenomenon that abnormal ratios of a pair of parental alleles is detected in the meiotic products. At the molecular level: the conversion of one gene’s sequence to that of another.

40. Fig

41. Fig

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

43. Fig

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

45. Fig

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.

51. Fig

52. Fig

53. Fig

54. Fig

55. Fig

56. Fig

57. Fig

58. Transposition
Transposition is mediated by transposable elements, or transposons.
Transposons of bacteria: IS (insertion sequences) contains only sequences required for transposition and proteins (transposases) that promote the process. Complex transposons contain genes in addition to those needed for transposition.
Transposition is characterized by duplication of direct repeats (5-9 bps in most cases) at target site.
Transposition, in some instances, may be mediated through a RNA intermediate (retrotransposons and non-LTR retrotransposons).

59. Duplication of the DNA sequence at a target site when a transposon is inserted

60. Fig. 23.1

61. Fig. 23.2

62. Fig. 17.3

64. Fig. 23.3

65. Fig. 23.4

66. Fig. 23.5

67. Replicative transposition is meidated
by a cointegrate intermediate.
Fig. 23.6

68. Fig. 23.7

69. Eukaryotic transposons
DNA transposons: (i) Ds and Ac of maize, (ii) Drosophila P elements.
Retrotransposons: (i) LTR retrotransposons (Ty element of yeast and copia of Drosophila). (ii) non-LTR retrotransposons (LINES, Alu, group II introns).

70. Ds and Ac of maize
Fig. 23.8

71. Fig. 23.9

72. Fig

73. Hybrid Dysgenesis
Fig F

74. Fig

75. Fig

76. Fig.23.19

77. Fig

78. Fig

79. Fig

80. Fig

81. Fig

83. Fig

84. Nonviral transposons: LINES
Fig

85. Fig

86. Fig

87. Fig

88. Group II introns: Retrohoming

89. DNA Repair
DNA damage may arise: (i) spontaneously, (ii) environmental exposure to mutagens, or (iii) cellular metabolism.
DNA damage may be classified as: (I) strand breaks, (ii) base loss (AP site), (iii) base damages, (iv) adducts, (v) cross-links, (vi) sugar damages, (vii) DNA-protein cross links.
DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death.

90. Fig

91. Fig

92. Fig

94. Methylataion and Mismatch Repair

95. Model for Mismatch Repair

98. Base-Excision Repair

99. FIGURE 16.12: Uracil is removed from DNA
FIGURE 16.13: Glycosylases remove bases

100. 16.5 Base Excision Repair Systems Require Glycosylases
FIGURE 16.14: Base removal triggers excision repair

101. Nucleotide-Excision Repair in E. coli and Humans

103. Alkylation of DNA by alkylating agents

104. Direct Repair: Photoreactivation by photolyase

105. O6-methyl G, if not repaired, may produce a mutation

106. Direct Repair: Reversal of O6 methyl G to G by methyltransferase

107. Direct repair of alkylated bases by AlkB.

108. Effect of DNA damage on replication: (i) coding lesions won’t interfere with replication but may produce mutation, (ii) non-coding lesions will interfere with replication and may lead to formation of daughter-strand gaps (DSG) or double-strand breaks (DSB).
DSG and DSB may be repaired by recombination process, to be discussed in the following section.

109. Models for recombinational DNA repair

110. Fig

112. Model for nonhomologous end-joining
Fig
Model for nonhomologous end-joining

113. Figure 16.25: NHEJ requires several reactions.

114. Fig