DNA Recombination in real life Chromosomal homologous recombination

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 11/22/2020 1

Recombination occurs between homologous chromosomes Sister chromatids Dad 11/22/2020 Mom 2

Crossing-over at DNA Level Watson Mom/ Mom Dad/ Dad 11/22/2020 Crick X 5

Holliday’s Recombination Model (proposed by Holliday in 1964) 1. Alignment 2. Nicking at one strand of each chromosome 3. Strand invasion (or exchange) 4. Formation of Holliday junction 5. Branch migration 11/22/2020 MCB Figure 12 -29 6

Holliday’s Recombination Model (cont’ed) A B C A’ a’ b’ B’ C’ c’ a b c Holliday Junction 6. Resolution MCB Figure 12 -29 11/22/2020 7

“Double-strand break” recombination model 1. Alignment 2. Double-strand break → (exonuclease) → singlestrand with 3’ end exposed 3. Single-strand invasion → DNA polymerase → Holliday intermediate (2 crossovers) 4. Branch migration 5. Resolution of Holliday intermediate 11/22/2020 8

“Double-strand break” recombination model 1. Alignment 2. Double-strand break → (exonuclease) → singlestrand with 3’ end exposed 3. Single-strand invasion → DNA polymerase → Holliday intermediate (2 crossovers) 4. Branch migration 5. Resolution of Holliday intermediate Which sets of products have gone through recombination? 11/22/2020 9

Enzymology of Homologous Recombination -- in E. coli -- 1. Rec. BCD 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. Rec. A -- recombinase catalyzes ATP-dependent DNA strand invasion 4. Ruv. A, Ruv. B, Ruv. C junction binding, branch migration, resolution 5. Rec. E F G J N O Q R T 11/22/2020 10

How is 3’ end exposed single-strand DNA generated in E. coli? by Rec. BCD? 11/22/2020 11

Rec. A polymer forms helical structure This is not a double helix DNA !! Rec. A homologues are found in all species. 11/22/2020 12

Model for strand exchange mediated by Rec. A in vitro assay 11/22/2020 13

E. coli Ruv. ABC in branch migration and resolution of Holliday junction 11/22/2020 MCB Figure 12 -35 14

What remains unanswered? chiasmata 11/22/2020 Holliday junction 15

When replication fork meets damaged DNA Replication stalled Recombinational repair 11/22/2020 Replication reinitiated ori-independent Pri. A, B, C; Dna. B, C, G, T 16 Replication restart primosome

site-specific recombination within the same DNA essential elements? 1. a specific recombinase (protein) 2. a corresponding recombination site (short, unique DNA 11/22/2020 sequence, 20 ~ 200 bp) 17

Site-specific recombination in λ phage between two circular DNA att. B/att. P – 15 bp 11/22/2020 18

Transposon is flanked by short, direct repeat sequence. How? Although transposons are widespread in all species, their transposition occur very infrequently direct replicative In bacteria, antibiotic resistance genes are carried in transposons 11/22/2020 19

co-integrate 11/22/2020 20

Recombination of the V and J gene segments of the human Ig. G kappa light chain 11/22/2020 21