DNA Recombination: Homologous Recombination
DNA recombination: homologous recombination
Sister chromosomes Homologous (chromatids) chromosomes
Centromeres
Recombination
Complex of Tetrad homologous Result of chromosomes recombination (crossing over) 3' 5' 5' 3' Double-strand 5' 3' cleavage 3' 5'
3' 5' 5' 3' Degradation 5' 3' of 5'-ends 3' 5'
D-loop 3' 3' 5' 5' 3' Invasion 5' 3' 3' 3' 5' Holliday junction 3' 5' 5' 3' DNA repair 5' 3' synthesis 3' 5' Heteroduplexes 3' 5' 5' 3' Branch 5' 3' migration 3' 5' The error-free repair of a double-strand break during replication
Double- strand break 3' 5' 5' 3' Double-strand 5' 3' cleavage 3' 5'
3' 5' 5' 3' Degradation 5' 3' of 5'-ends 3' 5'
D-loop 3' 3' 5' 5' 3' Invasion 5' 3' 3' 3' 5' Holliday junction 3' 5' 5' 3' DNA repair 5' 3' synthesis 3' 5' Heteroduplexes 3' 5' 5' 3' Branch 5' 3' migration 3' 5' Initial steps of homologous recombination in E. coli
RecBCD Recognition of the double- strand break
Degradation of 5'-ends
Recognition of RecA the single- strand 3' tail 3' Invasion and D-loop formation
RecA
D-loop Eukaryotic system Holliday junction
3' 5' 5' 3' 5' 3' 3' 5'
Isomerization
Holliday junction in the complex with ruvA Branch migration
ruvA ruvB Branch migration
5'
5' 5' 5'
5' 5' 5'
5'
5' 5'
5' 5' Resolution of Holliday junction
A' B' C' 3' 5' 5' 3' A B C 5' 3' 3' 5' a' b' c'
B' Resolvase b A' a c C' 3' 5' 3' 5' 5' 3' 5' 3' A a' c' C b'
B B'
b A' a c C' 3' 5' 3' 5' 5' 3' 5' 3' A a' c' C b'
B
A' b' c' A B c
a b C
a' B' C' B' 1 4 b A' a c C' 3' 5' 3' 5' 5' 3' 5' 3' A a' c' C b' 2 3 B Cuts 1+3 Cuts 1+4
Recombination No recombination
A' b' c' A' b' C'
A B c A B C a b C a b c a' B' C' a' B' c' DNA recombination: site-specific recombination
λ DNA
1st cleavage Integrase attP attB (Recombinase)
Bacterial DNA Strand exchange 2nd cleavage and ligation
Strand exchange, ligation Integration Recombinase is a site-specific topoisomerase I
1st cleavage and ligation
Tyr
Recombinase subunits
Isomerization of Holliday junction, 2nd cleavage
Ligation Recombinase
Insertion
Excision
Inversion Inversion in bacteriophage μ Cre dependent site-specific recombination in P1 bacteriophage Transposition of mobile (transposable, interspersed) DNA elements
Two classes of mobile elements
DNA transposon Retrotransposon
Transcription
“cut” RNA intermediate Reverse transcription
Insertion “paste” DNA transposons
Transposase binding
Synaptic complex formation
Cleavage
Target capture
Strand transfer Replication independent Transposition during transposition replication Transposon
The double-strand break is repaired by NHEJ-system
Exact repair by homologous recombination: the transposon is recovered LTR retroposons
Nucleus Cytoplasm Coding region LTR 5–8 kb Insertion
Transcription
mRNA
DNA Translation Reverse transcription
Integrase
Synthesized Reverse transcriptase and Integrase Retroviruses Nucleus Cytoplasm Coding region LTR 5–8 kb
Insertion DNA Transcription
mRNA Reverse transcription
Viral proteins Translation Virion
Integrase Infection
Reverse transcriptase Viral RNA
Viral / retroposon DNA
Integrase
Cellular DNA LINE (Long Interspersed Elements)
Nucleus Coding region ~5 kb Reverse transcription and insertion
Transcription
mRNA
Translation
Synthesized Reverse transcriptase and Integrase Cytoplasm SINE (Short Interspersed Elements)
Nucleus Non-coding DNA < 500 bp Reverse transcription and insertion
Transcription (RNA pol III)
RNA
Reverse transcriptase and Cytoplasm Integrase expressed from a LINE Non-homologous end joining: recombination that is independent on sequence Immunoglobulins
Antigen
Antigenic determinant Immunoglobulin V structure
V
C C CL domain VL domain VH domain
Antigen Antigen binding binding site site
Heavy chains
Light chains Immunoglobulin gene clusters
~ 100 ~ 30 6 H chain
V1 VX VN D1 DX DN J1 JX J6 C
~ 18 000 combinations
~ 100 4 L chain
V1 VX VN J1 JX J4 C
~ 400 combinations
~ 7 · 106 combinations in total V(D)J – recombination
V1 VX VN D1 DX DN J1 JX J6 C
V1 VX DX JX J6 C
Promoter Enhancer 1st step of V(D)J–recombination: site-specific recombination
RSS 23 12 12 23 V D J
CACAGTG- -ACAAAAACC -CACTGTG 23 bp GGTTTTTGT- 12 bp GTGTCAG- -TGTTTTTGG CCAAAAACA- -GTGACAC heptamer nonamer nonamer heptamer V 7 RAG 5' 9 7 D 12 23 V 7 9 RAG
23 9 7 7 9 12
RAG 1/2 are the products of two V D Recombination-Activating Genes V D
5' 3' 3' 5'
Deoxyribonucleotidyl transferase
5' 3' 3' 5'
NHEJ
Reparative synthesis