Enzymes required for recombination Overview Generation of single strands Invasion of single strands...

Enzymes required for recombination

Overview

Generation of single strands

Invasion of single strands

Branch migration

Resolution

Identification of enzymes required for recombination

• Screen bacteria and yeast for mutants with decreased recombination frequency – Isolate mutants defective in recombination

• Organize into >20 complementation groups• Gene names: recA, recB, recC, recD, …recJ, …• Purify the proteins encoded by these genes and

determine their enzymatic function• We still do not have a complete picture of how

these enzymes carry out all the steps in recombination.

Overview of enzymatic steps

• Pre-synaptic events – generate single-stranded DNA tails on duplex -

3 pathways for this in E. coli– RecA and SSB bind these single stranded tails

• Synaptic events – pairing and strand exchange (RecA protein)

• Post-synaptic events– branch migration (RuvA & RuvB)– isomerization & resolution (RuvC)

Overview of enzymatic steps in

recombination

Generation of single strands I• Recombination pathways studied in bacterial strains

with a high frequency of recombination (Hfr strains)• RecBCD pathway in w.t. cells

– recBCD- reduces frequency ~ 500-fold– also causes viability and DNA repair problems

• Suppressors that restore Rec+ phenotype– recBCD- sbcBC- enables RecF pathway

• Numerous genes encode enzymes in this pathway (recQ, recJ, recF, …)

• See damage inducible increase in recombination

– recBCD- sbcA- enables RecE pathway• Production of exo VIII from cryptic prophage• Also depends on some genes from RecF pathway

Generation of single strands II• RecBCD enzyme

– Helicase– Endonuclease (single-stranded DNA only)– ATPase– Exonuclease (mainly 3’ to 5’, minor 5’ to 3’)– Pathway that produces high frequency of recombination

on one side of Chi sites: • 5’ GCTGGTGG 3’ (shown as - note asymmetry

• RecQ helicase (RecF pathway)– Converts duplex to single strands– plus RecJ: makes 5’ to 3’ exonuclease active on single

stranded DNA

• RecE: 5’ to 3’ exonuclease (exo VIII)

Chi sites

• Hot spots for recombination in E. coli• Sequence is 5’-GCTGGTGG- 3’• Signal to RecBCD to generate a DNA end:

– Before the chi site, RecBCD is a helicase and an exonuclease (3’ to 5’, working on the “top” strand).

– At the chi site, the 3’ to 5’ exo stops.– After the chi site, RecBCD is still a helicase and also

activates the 5’ to 3’ exonuclease (working on the “bottom” strand)

• This process leaves the Chi site at the 3’ end of a single stranded DNA.

RecBCD

3’5’

D

CB5’3’

“downstream” “upstream”

3’

5’

5’3’

5’

5’3’

helicase3’ to 5’ exonucleaseATP hydrolysis

3’

5’

5’3’

At , attenuate3’-5’ exo

At , activate5’-3’ exo

Helicase,ATP hydrolysis

Generation of single strands III

• Other pathways for generating free 3’ ends:– RecE pathway

• exo VIII is 5’ to 3’ exonuclease that works directly on duplex DNA

– RecF pathway • RecQ helicase • RecQ plus RecJ: ssDNA 5’ to 3’ exo

Overview of enzymatic steps

• 3 pathways to generate single strands in E. coli

• RecA + SSB: pair homologous DNAs

• RecA: strand exchange (assimilation)

• RuvA + RuvB: branch migration– RecA and RecG can also do this

• RuvC + DNA ligase: resolution– RuvA can substitute for RuvC

Pairing and strand invasion: RecA protein

• RecA is a small protein (38 kDa) with multiple functions:– ATPase– Binds single-stranded DNA– Assimilates single-stranded DNA into a

homologous duplex DNA– Stimulates protease activity of LexA during SOS

• Used in all 3 pathways of recombination in E. coli

• Homologs are in yeast (Rad51 and Dmc1) and mouse (Rad51): highly conserved

3’ end generated by RecBCD can invade a homologous molecule

3’

5’3’

5’3’

+

RecA, ATP

Single strand assimilation

• 1. RecA polymerizes onto single-stranded DNA in the presence of ATP to form the presynaptic filament.– Polymerization starts at or near the 5’ end – 1 molecule of RecA covers 3 to 5 nucleotides

• 2. Presynaptic filament aligns with a homologous region in the duplex DNA– Form a paranemic joint (“invading strand” is not

yet intertwined with its complementary strand)

Single strand assimilation, cont’d

• 3. Strands exchange to form a plectonemic joint.– Invading strand is intertwined with its

complement.– Exchange is in a 5’ to 3’ direction relative to the

invading single strand.– ATP is hydrolyzed to ADP+Pi to dissociate

RecA (recall that RecA is an ATPase).– Strand synonymous with the invading strand is

displaced (coated with SSB if present).

Action of

RecA in

strand assimi-lation

RecA: Static view of 3-D structure

Green: alpha-helices Blue: peptide backboneBrown: beta-sheets small molecule: ADP

DNA binding

ADP

Interactionsin filament

In vitro assay for single strand assimilation

• Need free 3’ end complementary to the invading single strand to see strand exchange (assimilation) in vitro

• e.g. use a single stranded circle plus a homologous linear duplex– Circular single strand is coated with RecA-ATP

= presynaptic filament– Form a paranemic joint (first stage of synapsis)– Single stranded circle invades the linear duplex,

with hydrolysis of ATP and dissociation of RecA– At completion, have a nicked circle plus a linear

single strand (displaced strand).

Assay for single strand assimilation:

Single stranded

circle converted to duplex circle

Overview of enzymatic steps

• 3 pathways to generate single strands in E. coli

• RecA + SSB: pair homologous DNAs

• RecA: strand exchange (assimilation)

• RuvA + RuvB: branch migration

• RuvC + DNA ligase: resolution

RuvA-Holliday junction structure

Branch migrationRuvA:RecognizesHollidayjoint

RuvB:Helicase, Promotes Branch mi-gration

Eggleston & West,1996 Trends in Genetics12, 20-25

Resolution

RuvC binds to HollidayJunction (with RuvA and RuvB), cleaves symmetrically (in strands on opposites sides of the Holliday joint, opposite polarities).It can cut either pair of strands to accomplish vertical or horizontal resolution.

After cleavage, ligase joins the ends to finish resolution.

Eggleston & West,1996, Trends in Genetics 12, 20-25

5’

5’3’

3’

Vertical & horizontal resolution

or