DNA Recombination -- in real life --

1. DNA Recombination-- in real life -- Chromosomal homologous recombination a. during meiosis (減數分裂) in higher plants and animals b. bacterial gene recombination gene transfer via i. conjugation ii. transduction iii. transformation (?) c. phage gene recombination when host cell is infected by two phages with different genotypes

2. Recombination occurs between homologous chromosomes Sister chromatids Mom Dad

3. Homologous recombination seldom occurs in Mitosis 有絲分裂 Mom Dad 雙套染色體 （體細胞） DNA複製 同源染色體不配對 DNA 重組 發生機率低 一次染色體分離 同源染色體 各自獨立進行 雙套染色體 （體細胞）

4. It occurs FREQUENTLY in Meiosis 減數分裂 Mom Dad 雙套染色體 （體細胞） 複製 同源染色體配對 DNA 重組 第一次染色體分離 第二次染色體分離 單套染色體 （精子、卵）

5. Crossing-over at DNA Level Watson Mom/ Mom Crick X Dad/ Dad

6. Holliday’s Recombination Model(proposed by Holliday in 1964) • Alignment • Nicking at one strand of each chromosome • Strand invasion (or exchange) • Formation of Holliday junction • Branch migration MCB Figure 12-29

7. Holliday’s Recombination Model (cont’ed) A B C A’ b’ C’ a’ B’ c’ a b c Holliday Junction 6. Resolution MCB Figure 12-29

8. “Double-strand break” recombination model • Alignment • Double-strand break → (exonuclease) → single-strand with 3’ end exposed • Single-strand invasion → DNA polymerase → Holliday intermediate (2 crossovers) • Branch migration • Resolution of Holliday intermediate

9. “Double-strand break” recombination model • Alignment • Double-strand break → (exonuclease) → single-strand with 3’ end exposed • Single-strand invasion → DNA polymerase → Holliday intermediate (2 crossovers) • Branch migration • Resolution of Holliday intermediate Which sets of products have gone through recombination?

10. Enzymology of Homologous Recombination-- in E. coli -- 1. RecBCD complex helicase activity: ATP-dependent unwinding nuclease activity: cleaves SS DNA 2. SSB: single-stranded DNA binding protein protects SS DNA from cleavage by nuclease 3. RecA -- recombinase catalyzes ATP-dependent DNA strand invasion 4. RuvA, RuvB, RuvC junction binding, branch migration, resolution 5. RecE F G J N O Q R T

11. How is 3’ end exposed single-strand DNA generated in E. coli? by RecBCD?

12. RecA polymer forms helical structure This is not a double helix DNA !! RecA homologues are found in all species.

13. Model for strand exchange mediated by RecA in vitro assay

14. E. coli RuvABC in branch migration and resolution of Holliday junction MCB Figure 12-35

15. What remains unanswered? Holliday junction chiasmata

16. When replication fork meets damaged DNA Replication stalled Recombinational repair Replication reinitiated ori-independent PriA, B, C; DnaB, C, G, T Replication restart primosome

17. site-specific recombinationwithin the same DNA • essential elements? • a specific recombinase (protein) • a corresponding recombination site (short, unique DNA sequence, 20 ~ 200 bp)

18. Site-specific recombination in λ phagebetween two circular DNA attB/attP – 15 bp

19. Transposon is flanked by short, direct repeat sequence. How? Although transposons are widespread in all species, their transposition occur very infrequently In bacteria, antibiotic resistance genes are carried in transposons replicative direct

20. co-integrate

21. Recombination of the V and J gene segments of the human IgG kappa light chain