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
November 23, 2018

2. Recombination occurs between homologous chromosomes
Sister chromatids
Dad
Mom
November 23, 2018

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）
November 23, 2018

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）
November 23, 2018

5. X Crossing-over at DNA Level Mom/ Mom Dad/ Dad Watson Crick
November 23, 2018

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
November 23, 2018

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
November 23, 2018

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
November 23, 2018

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?
November 23, 2018

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
November 23, 2018

11. How is 3’ end exposed single-strand DNA generated in E. coli?
by RecBCD?
November 23, 2018

12. RecA polymer forms helical structure
This is not a double helix DNA !!
RecA homologues are found in all species.
November 23, 2018

13. Model for strand exchange mediated by RecA
in vitro assay
November 23, 2018

14. E. coli RuvABC in branch migration and resolution of Holliday junction
November 23, 2018
MCB Figure 12-35

15. What remains unanswered?
chiasmata
Holliday junction
November 23, 2018

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
November 23, 2018

17. site-specific recombination within the same DNA
essential elements?
a specific recombinase (protein)
a corresponding recombination site (short, unique DNA sequence, 20 ~ 200 bp)
November 23, 2018

18. Site-specific recombination in λ phage between two circular DNA
attB/attP – 15 bp
November 23, 2018

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
direct
replicative
November 23, 2018

20. co-integrate
November 23, 2018

21. Recombination of the V and J gene segments of the human IgG kappa light chain
November 23, 2018