Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 22
Homologous
Recombination
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
22.1 Homologous
Recombination Pathways
22-2
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
22-3
The RecBCD Pathway Schematic
RecBCD pathway is a well-studied homologous
recombination pathway used by E. coli
22-4
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
22-5
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
22-6
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
22-7
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-DNA
2. Synapsis – alignment of complementary sequences
in ss- and ds-DNAs
3. Postsynapsis – ss-DNA replaces the (+) strand in
ds-DNA to form a new double helix
– Joint molecule is an intermediate in this process
22-8
Presynapsis
In 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
22-9
Synapsis:
• Synapsis is the proper
alignment of
complementary
sequences
• Synapsis occurs when:
– Single-stranded DNA
finds a homologous
region in a doublestranded DNA
– This ss-DNA aligns with
the ds-DNA
• No intertwining of the 2
DNAs occurs at this
point
22-10
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
22-11
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 ssDNA ends that RecA needs to initiate strand
exchange
22-12
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
22-13
A Synthetic Holliday Junction
• Mix oligonucleotides at
annealing conditions
for complementary
base-pairing
• 5’-end of oligo 2 basepairs with the 3’-end of
oligo 1
• 5’-end of oligo 1 basepairs with the 3’-end of
oligo 2
• Ends cross over in
pairing
22-14
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
22-15
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
22-16
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
22-17
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
22-18
Model of Yeast Recombination
22-19
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
22-20
DSB End Resection
• Resection occurs on both
strands using prior nicks
• Recombinases load
asymmetrically onto the
newly created singlestranded regions
• One protein tags coated
free 3’-end for invasion
into homologous duplex
• This leads to initiating
Holliday complex
formation
22-21
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
22-22
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
22-23
Gene Conversion Model
• Strand exchange event with branch migration
during sporulation has resolved to yield 2 duplex
DNAs with patches of heteroduplex
22-24
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
22-25