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

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

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

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

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

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

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

“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

“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

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

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

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

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

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

What remains unanswered? chiasmata Holliday junction November 23, 2018

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

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

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

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

co-integrate November 23, 2018

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