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Molecular BiologyFourth Edition
Chapter 22
Homologous Recombination
Lecture PowerPoint to accompany
Robert F. Weaver
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
22-2
22.1 Homologous Recombination Pathways
22-3
RecBCD Pathway – Initial Binding
RecBDC-sponsored homologous recombination in E. coli:
– DNA helicase activity unwinds the DNA toward a Chi-site
• Sequence 5’-GCTGGTGG-3’• Chi sites found on average every 5000 bp in E. coli
genome
– RecBCD protein has • ds- and ss-exonuclease activity• ss-endonuclease activity• Activities permit RecBCD to produce a ss-tail now
coated by RecA protein
22-4
The RecBCD Pathway Schematic
RecBCD pathway is a well-studied homologous recombination pathway used by E. coli
22-5
RecBCD Pathway – D Loop
• Invasion of a duplex DNA by a RecA-coated single-stranded DNA from another duplex that has suffered a double-stranded break
• Invading strand forms a D loop (displacement)– Loop is defined by displaced DNA strand– When tail finds homologous region, nick occurs in in
D-looped DNA– Nick allows RecA and ss-break create a new tail that
can pair with gap in the other DNA
• Subsequent degradation of the D-loop strand leads to the formation of a branched intermediate
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Holliday Junctions
• Branch migration in this intermediate yields a Holliday junction with 2 strands exchanging between homologous chromosomes
• Branch in the Holliday junction can migrate in either direction by breaking old base pairs and forming new ones in a process called branch migration
• This migration process does not occur at a useful rate spontaneously– DNA unwinding required– Unwinding requires helicase activity and energy from
ATP
22-7
Resolving Holliday Junctions
• Holliday junctions can be resolved by nicking 2 of its strands
• Yielding: – 2 noncrossover recombinant DNAs with
patches of heteroduplex– 2 crossover recombinant DNAs that have
traded flanking DNA regions
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22.2 Experimental Support for the RecBCD Pathway - RecA
• The recA gene has been cloned and overexpressed with abundant RecA protein available for study
• It is a 38-kD protein that can promote a variety of strand exchange reactions
• There are 3 stages of participation of RecA in strand exchange
1. Presynapsis – RecA coats the ss-DNA2. Synapsis – alignment of complementary sequences
in ss- and ds-DNAs3. Postsynapsis – ss-DNA replaces the (+) strand in
ds-DNA to form a new double helix– Joint molecule is an intermediate in this process
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PresynapsisIn the presynapsis step of recombination:
– RecA coats a ss-DNA participating in recombination
– SSB accelerates the recombination process• Melting secondary structure• Preventing RecA from trapping any secondary
structure that would inhibit strand exchange later in the recombination process
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Synapsis:• Synapsis is the proper
alignment of complementary sequences
• Synapsis occurs when:– Single-stranded DNA
finds a homologous region in a double-stranded DNA
– This ss-DNA aligns with the ds-DNA
• No intertwining of the 2 DNAs occurs at this point
22-11
Postsynapsis:Strand Exchange
• RecA and ATP collaborate to promote strand exchange between ss- and ds-DNA
• ATP is necessary to clear RecA off the synapsing DNAs
• This makes way for formation of ds-DNA involving the single strand and one of the strands of the DNA duplex
22-12
RecBCD
• RecBCD has a DNA endonuclease activity– Nicks ds-DNA especially near Chi sites– ATPase-driven DNA helicase activity that can
unwind ds-DNA from their ends– The activities help RecBCD provide the ss-
DNA ends that RecA needs to initiate strand exchange
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RuvA and RuvB• RuvA and RuvB form a DNA helicase that can
drive branch migration• RuvA tetramer with square planar symmetry
recognizes the center of a Holliday junction and binds to it
• Likely induces the Holliday junction itself:– To adopt a square planar conformation– To promote binding of hexamer rings of RuvB to 2
diametrically opposed branches of the Holliday junction
• RuvB uses its ATPase to drive the DNA unwinding and rewinding necessary for branch migration
22-14
A Synthetic Holliday Junction
• Mix oligonucleotides at annealing conditions for complementary base-pairing
• 5’-end of oligo 2 base-pairs with the 3’-end of oligo 1
• 5’-end of oligo 1 base-pairs with the 3’-end of oligo 2
• Ends cross over in pairing
22-15
RuvC
• Resolution of Holliday junctions is catalyzed by the RuvC resolvase– This protein acts as a dimer to clip 2 DNA strands to
yield either patch or splice recombinant products– Clipping occurs preferentially at the consensus
sequence 5’-(A/T)TT(G/C)-3’
• Branch migration is essential for efficient resolution of Holliday junctions– Essential to reach preferred cutting sites– RuvA, B, and C work together in a complex to locate
and cut those sites
22-16
Resolution of a Holliday Junction
Holliday junction can be resolved in 2 ways:• Cuts 1 and 2 yield 2 duplex DNAs with patches of
heteroduplex • Cuts 3 and 4 yield crossover recombinant molecules with
the 2 parts joined by a staggered splice
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22.3 Meiotic Recombination
• Meiosis in most eukaryotes is accompanied by recombination
• This process shares many characteristics with homologous recombination in bacteria
• This section focuses on meiotic recombination in yeast
22-18
Mechanism Overview
• Start with chromosomal lesion: ds-DNA break• Next exonuclease recognizes the break
– Digests the 5’-end of the 2 strands– Creates 3’-single strand overhangs
• One single-stranded end can invade other DNA duplex, forming a D loop
• DNA repair synthesis fills in the gaps in the top duplex expanding the D loop
• Branch migration can occur in both directions leading to 2 Holliday junctions
• Holliday junctions can be resolved to yield either a noncrossover or a crossover recombinant
22-19
Model of Yeast Recombination
22-20
The Double-Stranded DNA Break
• DNA cleavage uses 2 Spo11– Active site Tyr as OH– Attack 2 DNA strands at
offset positions– Transesterification reaction
breaks phosphodiester bonds within DNA strands
– Creates new bonds
• Nicking DNA strands– Nicking is asymmetric– Yields 2 sizes oligos
• Release of Spo11-linked oligos 12-37 nt long
22-21
DSB End Resection
• Resection occurs on both strands using prior nicks
• Recombinases load asymmetrically onto the newly created single-stranded regions
• One protein tags coated free 3’-end for invasion into homologous duplex
• This leads to initiating Holliday complex formation
22-22
Creation of Single-Stranded Ends at DSBs
• Formation of the DSB in meiotic recombination is followed by 5’3’ exonuclease digestion of the 5’-ends at the break
• Digestion yields overhanging 3’-ends that can invade another DNA duplex
• Rad50 and Mre11 collaborate to carry out this reaction
22-23
22.4 Gene Conversion
• When 2 similar, non-identical DNA sequences interact, possibility exists for gene conversion– Conversion of one DNA sequence into that of
another
• Sequences participating in gene conversions can be:– Alleles, as in meiosis– Nonallelic genes, such as the MAT genes that
determine mating type in yeast
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Gene Conversion Model
• Strand exchange event with branch migration during sporulation has resolved to yield 2 duplex DNAs with patches of heteroduplex
22-25
Gene Conversion Without Mismatch Repair
• Consider from the middle of the DSB recombination scheme
• Invading strand is partially resected
• DNA repair synthesis more extensive
• Branch migration and resolution do not change nature of the 4 DNA strands
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