DNA Recombination Biochemistry 302

February 1, 2006

Genetic recombination: Why is it advantageous to a cell? •

Bacterial cell – Information exchange during bacterial conjugation (mating) between donor F+ and recipient F- cells – Recombinatorial repair: reconstruction of replication forks stalled at the site of DNA damage using non-mutated template strand

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Eukaryotic cells – Maintaining genetic diversity: Information exchange (crossing over) between paired sister chromatids in germ cells during meiotic prophase I – Recombinatorial repair (dsDNA breaks, stalled replication forks) – Chromosome length maintenance in cells lacking telomerase; orderly chromosome segregation during 1st meiotic cell division – Programmed genetic rearrangements during development (Ig genes) Lehninger Principles of Biochemistry, 4th ed., Ch 25

Classes of recombination processes Table 25-3

Homologous recombination is stimulated by processes that break DNA strands (e.g. ionizing UV). Lehninger Principles of Biochemistry, 4th ed., Ch 25

Holliday Model (two strand break) Nicking

– Nicking at same location on aligned homologous strands of two chromosomes

Strand invasion

– Partial unwinding then strand invasion of other duplex – Nick ligation to generate a cross-strand intermediate: Holliday junction

Ligation

– Branch migration (moves by bp exchange) – “Resolution” of junction: Isomerization → strand breaking & rejoining yields two possible outcomes: nonrecombinant or recombinant heteroduplexes (different strands from those 1st nicked)

Branch migration

5

Parallel geometry

3

3

Resolution Antiparallel geometry

5

5 3

3 5

Fig. 25-20

Different version of double-strand break model Alignment & ds break Extension & branch migration ds gap

ssDNA extension

strand invasion

DNA synthesis or gap repair

Resolution

Recombinant Lehninger Principles of Biochemistry, 4th ed., Ch 25

Anatomy of a three-stranded branch point and Holliday intermediate

Two bacterial plasmids Lehninger Principles of Biochemistry, 4th ed., Ch 25

Core interactions at junction crossover (common A6C7C8 or R6-C7-Y8 motif)

parallel stacked-X

WC H-bonds red dots ACC H-bonds blue dots

open-X (+ protein)

antiparallel stacked-X

Na+ cavity formed by two A6 phosphates and bases of A6 and C7 nucleotides

B.F. Eichman et al. (2000) PNAS 97:3971 F.A. Hays et al. (2003) JBC 278:49663…structure of d(CCGGTACCGG) as a junction

Proteins involved in homologous recombination in E. coli • Phase1: Initiation/exposure of ssDNA end – RecBCD enzyme: Trimeric complex possesses both helicase and nuclease activity (ATP-dependent). Binds free or broken end of DNA and moves inward. – Encoded by recB, recC, recD genes – Some other nucleases and RecQ helicase

• Phase 2: Homologous pairing/strand exchange – RecA: the major player in strand exchange – SSB, RecF, RecO, RecR (RecFOR regulate RecA filament assembly and disassembly)

• Phase III: Branch migration/junction resolution – RuvAB (repair of UV damage): Motor complex binds Holliday intermediates, displaces RecA, promotes branch migration (faster than RecA). – RuvC: A “resolvase” which cleaves Holliday intermediates

RecBCD initiation complex exposes 3′ single-stranded ends • •

Initial binding site: doublestrand break Chi (crossover hotspot instigator) 5′GCTGGTGG3′ – 1009 in E. coli genome – Enhance frequency of recombination ~5-10-fold within 1000 bp of chi – Suppress nuclease activity of RecBCD but in a strandspecific manner

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Single-stranded 3′ serves as recognition site for RecA filament assembly Lehninger Principles of Biochemistry, 4th ed., Ch 25

Biochemical properties of RecA (Rad51) • Structure/Function – 38 kDa protein – Binds cooperatively to ssDNA as a homopolymer to form ordered helical filament (6 subunits/turn, length up to several thousand monomers) – Binds to single-stranded gap in duplex DNA – DNA-dependent ATPase

RecA-ssDNA nucleoprotein filament

• Role in recombination – Promotes pairing of homologous strands – Promotes 3 strand exchange process – Requires a free ssDNA end and DNA-DNA homology for strand invasion Contour model: Three-strand intermediate

Biochemical and mechanistic features of RecA-mediated DNA strand exchange •RecA encircles ssDNA as a polymeric right-handed helical filament starting on 3′end. •RecA-ssDNA filament (w/ATP) takes up homologous dsDNA & aligns it with complementary sequence in ssDNA. •DNA underwinds & stretches to 1.5x its normal length to form a joint/hybrid molecule. •Once 3-strand intermediate forms, strand exchange can occur (w/ ATP hydrolysis), rate Fig. 25-24 ~6 bp/s in 5′→3′ direction.

RecA-ssDNA filament

Joint/hybrid molecule need ATP

Strand exchange need ATP

•Strand exchange is catalyzed by RecA in vitro, but additional proteins are needed in vivo.

Models for strand exchange induced by RecA in vitro

Because DNA is a helical structure, continued strand exchange requires an ordered rotation of the two aligned DNAs.

Lehninger Principles of Biochemistry, 4th ed., Ch 25

Other proteins assist in branch migration and resolution of Holliday junctions • RuvA – DNA-binding protein with specificity for four-strand Holliday junction (tetramer) – RuvB binding protein

• RuvB – ATP-dependent motor protein/pump (hexamer) – Binds two junction “arms” – Facilitates branch migration via DNA spooling

• RuvC – Resolvase (endonuclease) Fig. 25-29 – Initiates resolution of Holliday junction by nicking/breaking of two strands)

Twin pump model

Homologous recombination and repair of stalled replication forks • Daughter strand gap – RecA-mediated – “Good” template strand provides information to fill gap at replication fork.

• Origin-independent replication restart – Replication restart primasome (PriA, B, C & DnaB, C, G, T) – DNA polymerase II

• Double-strand break (eukaryotes) – Rad51, BRCA1?, BRCA2 – RuvA, RuvB-like proteins

Fig. 25-15

BRCA2 enhances RAD51-dependent strand exchange in vitro

Joint molecule

Nicked circle φX174 dsDNA

φX174 ssDNA Fig. 25-30

Yang, H. et al. (2002) Science 297:1837

Essential role of eukaryotic mediators in presynaptic filament assembly

(slow)

P. Sung et al. (2003) JBC 278:42729

Features of site-specific recombination •

Protein-DNA dependent – No requirement for DNA homology per se or high energy cofactors (e.g. ATP) but need “alignment” factors – Exchange occurs between short (often complementary) cis-acting recombination sites (~20-200 bp) – No new DNA synthesis, recombination sites regenerated when reaction is complete

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Mechanism of site-specific recombination: Note active site tyrosine residue forms transient phosphotyrosine linkage.

“Recombinase” required – Responsible for cutting and ligating at specific sites – Sight-specific endonuclease and ligase in one package – Promotes Holliday intermediate formation (integrase-class recombinases e.g Cre, λ phage integrase) – Intermolecular or intramolecular mechanisms (inversion, insertion, or deletion)

Surface contour model of Cre recombinase (four subunits) Lehninger Principles of Biochemistry, 4th ed., Ch 25

Example of site-specific recombination λ phage lysogeny

Excision of prophage (Int, IHF, and Xis) DNAbending factors

Fig. 25-17

Integration assisted by a host bacterial cofactor protein, IHF

Formation of intasome requires combined action of IHF and integrase attP:230 bp attB:23 bp 15 bp overlap

Proline intercalation between bps

Fig. 25-31

P.A. Rice et al. Cell 87:1295, 1996 Integration Host Factor DNA complex: Note how DNA is bent >160° to facilitate integrase binding to adjacent sites in λ att (7 Int/230 bp attP)