84 221 The RecBCD Pathway for Homologous Recombination

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Chapter 22 / Homologous Recombination
of fertilized human eggs are aneuploid; that is, they contain an abnormal number of chromosomes, which is usually a lethal problem. And one of the leading causes of
this aneuploidy is a reduction in the number, or abnormal
placement, of recombination events during meiosis. Also,
as we saw in Chapter 20, homologous recombination
plays an important role in allowing cells to deal with DNA
damage by so-called recombination repair.
Figure 22.1 illustrates several variations on the theme
of homologous recombination. Each variation is characterized by a crossover event that joins DNA segments
that were previously separated. This does not mean the
two segments must start out on separate DNA molecules. Recombination can be intramolecular, in which
case crossover between two sites on the same chromosome either removes or inverts the DNA segment in between. On the other hand, bimolecular recombination
involves crossover between two independent DNA molecules. Ordinarily, recombination is reciprocal—a twoway street in which the two participants trade DNA
segments. DNA molecules can undergo one crossover
event, or two, or more, and the number of events strongly
influences the nature of the final products.
22.1 The RecBCD Pathway for
Homologous Recombination
To illustrate the principles of homologous recombination,
let us consider the well-studied RecBCD pathway, one of
the homologous recombination pathways used by E. coli.
This recombination process (Figure 22.2) begins with
the induction of a double-stranded break in one of the recombining DNAs. The RecBCD protein, the product of the
recB, –C, and –D genes, binds to a DNA double-stranded
break and uses its DNA helicase activity to unwind the
DNA toward a so-called Chi site or x (Chi 5 crossover
hotspot instigator), which has the sequence 59-GCTGGTGG-39. Chi sites are found on average every 5000 bp in
the E. coli genome. RecBCD also has double-stranded and
single-stranded exonuclease and single-stranded endonuclease activities. These allow RecBCD to produce a singlestranded tail, which can then be coated by RecA protein
(the product of the recA gene). RecBCD also helps load
RecA onto the 39-DNA tail.
RecA allows the tail to invade a double-stranded DNA
duplex and search for a region of homology. This creates a
displacement loop (D-loop), defined by the displaced DNA
strand. Once the tail finds a homologous region, a nick
occurs in the D-looped DNA, possibly with the aid of
RecBCD. This nick allows RecA and SSB to create a new
tail that can pair with the gap in the other DNA. DNA
Intermolecular:
(a) Single crossover:
(b) Double crossover:
Intramolecular:
(a) Direct repeats:
(b) Inverted repeats:
Figure 22.1 Examples of recombination. The X’s represent crossover events between the two chromosomes or parts of the same chromosome.
To visualize how these work, look at the intermediate form of the reciprocal recombination on the top line. Imagine the DNAs breaking and forming
new, interstrand bonds as indicated by the arms of the X. This same principle applies to all the examples shown.
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22.1 The RecBCD Pathway for Homologous Recombination
3′
5′
Figure 22.2 The RecBCD pathway of homologous
recombination. (a) The RecBCD protein (omitted for the sake of
clarity) binds at a double-stranded DNA break, and the DNA helicase
activity of RecBCD then unwinds the DNA toward a Chi site, ultimately
creating a 39-terminal, single-stranded DNA that is coated with RecA
protein (yellow spheres) (b) RecA promotes invasion of another DNA
duplex, forming a D-loop. (c) RecA helps the invading strand scan for
a region of homology in the recipient DNA duplex. Here, the invading
strand has base-paired with a homologous region, releasing RecA.
(d) Once a homologous region is found, a nick in the looped-out DNA
appears, perhaps caused by RecBCD. This permits the tail of the
newly nicked DNA to base-pair with the single-stranded region in the
other DNA, probably aided by RecA. (e) The remaining gaps are filled
in and nicks are sealed by DNA ligase, yielding a four-stranded
complex with a Holliday junction. (f) Branch migration occurs,
sponsored by RuvA and RuvB. Notice that the branch has migrated to
the right. (g and h) Nicking by RuvC resolves the structure into two
molecules, crossover recombinants or heteroduplexes, respectively.
5′
3′
Chi
(a)
RecBCD unwinds DNA and leaves
3′ – protruding end, coated with RecA
(b)
Strand invasion; D-loop formation
(c)
Scan for homology.
3′
5′
3′
(d)
Nick
Strand exchange (RecA + SSB)
(e)
3′
5′
Repair gaps and seal nicks
(Holliday junction)
5′
3′
5′
3′
3′
5′
(f)
Branch migration (RuvA + RuvB)
(g)
Crossover resolution (RuvC)
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Branch migration does not occur at a useful rate spontaneously. Just as in DNA replication, DNA unwinding is
required, and this in turn requires helicase activity and energy from ATP. Two proteins, RuvA and RuvB, collaborate
in this function. Both have DNA helicase activity, and RuvB
is an ATPase, so it can harvest energy from ATP for the
branch migration process. Finally, two DNA strands must
be nicked to resolve each Holliday junction into heteroduplexes or recombinant products. The RuvC protein carries
out this function. Two alternative products can be produced, depending on which strands are nicked by RuvC.
If the inner strands of the Holliday junction are nicked
(Figure 22.3a), the structure resolves into a noncrossover
recombinant, also known as a patch recombinant, or heteroduplex. If the outer strands of the Holliday junction are
nicked (Figure 22.3b), the structure resolves into a crossover recombinant, also known as a splice recombinant, in
which the DNA duplex changes from one genotype (represented by blue) at one end to another (represented by red)
at the other.
SUMMARY RecBCD-sponsored homologous re-
(h) Noncrossover resolution (RuvC)
ligase seals both nicks to generate a Holliday junction,
named for Robin Holliday, who first proposed them in
1964. Holiday junctions are also known as half chiasmas
and Chi structures. The branch in the Holliday junction
can migrate in either direction simply by breaking old base
pairs and forming new ones in a process called branch
migration.
combination in E. coli begins with invasion of a
duplex DNA by a RecA-coated single-stranded
DNA from another duplex that has suffered a
double-stranded break. The invading strand forms a
D-loop. Subsequent degradation of the D-loop strand
leads to the formation of a branched intermediate.
Branch migration in this intermediate yields a Holliday junction with two strands exchanging between
homologous chromosomes. Finally, the Holliday
junction can be resolved by nicking two of its
strands. This can yield two noncrossover recombinant DNAs with patches of heteroduplex, or two
crossover recombinant DNAs that have traded
flanking DNA regions.