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 c) - 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
c
“downstream”
5’
3’
ATP hydrolysis
c
Helicase,
ATP hydrolysis
5’
3’
At c, attenuate
3’-5’ exo
At c, activate
5’-3’ exo
3’
5’
helicase
3’ to 5’ exonuclease
5’
3’
5’
3’
“upstream”
3’
5’
c
c
3’
5’
5’
D
BC
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
c
5’
3’
3’
+
RecA, ATP
5’
3’
c
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
assimilation
RecA: Static view of 3-D structure
DNA binding
Interactions
in filament
ADP
Green: alpha-helices
Brown: beta-sheets
Blue: peptide backbone
small molecule: ADP
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 migration
RuvA:
Recognizes
Holliday
joint
RuvB:
Helicase,
Promotes
Branch migration
Eggleston &
West,
1996 Trends in
Genetics
12, 20-25
Resolution
5’
3’
3’
5’
Eggleston & West,1996,
Trends in Genetics 12, 20-25
RuvC binds to Holliday
Junction (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.
Vertical & horizontal resolution
or