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DNA Recombination: Homologous Recombination

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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.
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