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.

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22.1 Homologous Recombination Pathways

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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

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The RecBCD Pathway Schematic

RecBCD pathway is a well-studied homologous recombination pathway used by E. coli

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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

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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

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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

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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

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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

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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

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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

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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

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Model of Yeast Recombination

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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

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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

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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

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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

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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