In the previous sections of this chapter, we have discussed how the genome is
faithfully reproduced from one generation to another during DNA replication and how
the correct sequence is maintained by DNA-repair processes throughout the life of a
cell and organism. In this section, we examine the mechanisms by which the genome
can change to generate new combinations of genes.
Soon after Mendel’s rules of independent gene segregation were rediscovered
and the segregation of linked groups of genes on individual chromosomes was widely
recognized, another great genetic discovery was made in D.
melanogaster: blocks of genes from homologous chromosomes could be
exchanged by the process of crossing over, or homologous recombination (often
referred to simply as recombination).
Homologous recombination takes place during meiosis in sexually reproducing
organisms. Recall that each homologous paternal and maternal chromosome contains a
different combination of alleles. By generating new chromosomes that contain part of
each homologous paternal and maternal chromosome, recombination results in new
combinations of alleles on a given chromosome. Thus recombination provides a
mechanism for generating genetic diversity beyond that achieved by the independent
segregation of chromosomes (see Figure 8-3).
Genetic exchange by recombination occurs not only in animals and plants but also in
prokaryotes, viruses, plasmids, and even in the DNA of cell organelles such as
mitochondria.
The events in a reciprocal recombination are equivalent to the
breakage of two homologous duplex DNA molecules, an exchange of
both strands at the break, and a resolution of the two duplexes
so that no tangles remain. To a good approximation, recombination occurs randomly
between two homologous DNA segments, and thus the frequency of recombination between
two sites is proportional to the distance between the sites. (As discussed in Chapter 8, this phenomenon is the basis
of genetic mapping of genes defined by mutations.) In the remainder of this chapter,
we describe how several types of proteins catalyze steps in recombination.
The Crossed-Strand Holliday Structure Is an Intermediate in
Recombination
Figure 12-29
.
Holliday model of genetic recombination
Genetically distinct homologous chromosomes (i.e., double-stranded
DNA molecules) are indicated by red and blue; alleles are indicated
by capital and lowercase letters (A, a).
Complementary DNA strands are distinguished by darker and lighter
shades and by the presence or absence of prime signs (A,
A′; a,
a′). Resolution of the
crossed-strand Holliday structure could occur by two different
pathways. Steps 4 and 5a or 6
, 7a , and 8a would yield spliced products
that exhibit recombination from AC/ac to
Ac/aC, with heteroduplex DNA containing the
B locus in between. Although step
5b or steps 7b and 8b
also resolve the connected strands of the Holliday structure, the
resulting patched products are not recombinants, since all markers
surrounding the crossover site, that is, to the left of
A and to the right of C, are
derived from the same initial chromosome. However, these molecules
are heteroduplex for the B segment, and as each of
these DNA molecules is duplicated, half the progeny will have the
B genetic marker and half will have
b. [Steps
1 – 5, see R. Holliday, 1964,
Genet. Res.
5:282; steps 6, 7a, 7b, 8a, and 8b see D. Dressler and
H. Potter, 1982, Ann. Rev. Biochem.
51:727; also see M. Meselson and C. M. Radding, 1975,
Proc. Nat’l Acad. Sci. USA
72:358; and N. Sigal and B. Alberts, 1972, J.
Mol. Biol.
71:769.]
In 1964, Robin Holliday proposed the
recombination model depicted in . Except for the initial
steps, this model appears to accurately describe the molecular events that lead
to genetic
recombination. According to the original Holliday model, after two
homologous double-stranded DNA molecules (i.e., cellular or viral
chromosomes)
become aligned, a nick is made in one strand of each of the recombining DNAs
(step 1). The two nicked strands then invade each other, a process called
strand exchange, at the site of the nicks, and the cut
3′ ends are joined to the 5′ ends of the homologous strand,
producing a crossed-strand
Holliday
structure (step 2). The branch point then migrates, creating a
heteroduplex region containing one
strand from each parental DNA molecule (step 3).
Two mechanisms have been proposed for separation, or
resolution,
of the connected duplexes. According to Holliday’s original proposal,
all four strands are cut at the crossover site (, step 4). If the left side of
chromosome I joins the
right side of
chromosome II, and vice versa (step 5a), then both strands in each
of the resulting duplexes are
recombinant; that is, all markers
to the left and right of the crossover site have undergone reciprocal
recombination. However, over a short
region — the
B and
b genes in the figure — the
chromosomes are termed
heteroduplex; they have genetic material
from one
chromosome on one strand and material from the other
chromosome on the
opposite strand. As each of these
chromosomes duplicates, half the progeny will
have the genetic marker
B from one initial
chromosome, and half
will have the
b allele. In contrast, if the left sides of
chromosomes I and II rejoin with their own respective right sides (step 5 b),
then both strands in each of the resulting duplexes are termed
nonrecombinant; that is, all markers to the left and right
of the crossover site are derived from the same initial
chromosome. However,
these molecules are also
heteroduplex for the
B or
b segment.
A later proposal simplifies the enzymatic cutting that is necessary to resolve
the crossed-strand intermediate. Rotation of the Holliday structure at the
crossover site forms a rotational isomer, or isomeric Holliday structure (step
6). (This can be visualized by imagining that the dotted line passing through
the crossover point in step 3 serves as an axis around which the bottom duplex
spins 180°, untwisting the crossed strands in the process.) Note that no
strands are cut or ligated in going from 3 to 6. The two connected duplexes of
this structure can be resolved (i.e., disconnected) by cutting and rejoining of
only two strands. If this involves the two strands that were not cut to generate
the original Holliday intermediate, the resulting “spliced”
products are recombinant duplex chromosomes containing a heteroduplex region
(steps 7a and 8a). However, if resolution involves cutting of the two strands
that were originally cut, the resulting “patched” products
are duplex chromosomes that contain a heteroduplex B region but
are not termed recombinants (step 7b and 8b), since all the markers to the left
of A and to the right of C are derived from
the same original chromosome.
Figure 12-30
.
Electron micrographs of plasmid DNA in the process of
recombination
(a) Circular plasmid DNA in crossed-strand Holliday structure. (b)
More highly magnified view reveals single-stranded ring in center of
isomeric Holliday structure that results from rotation about the
crossover point. [See H. Potter and D. Dressler, 1978, Cold
Spring Harbor Symp. Quant. Biol.
43:969; courtesy of D. Dressler.]
Evidence that Holliday intermediates actually exist has come from electron
microscopy of viral and
plasmid DNA molecules extracted from both bacterial and
animal cells. Electron micrographs of such molecules in the act of recombining
have revealed structures similar to the crossed-strand and isomeric Holliday
structures (). Thus,
regardless of the mechanism initiating
recombination, the process seems to
involve the kinds of intermediates predicted by the Holliday model.
Double-Strand Breaks in DNA Initiate Recombination
Many variations of the Holliday model have been proposed; these differ in how the
DNA strands are cut to initiate strand exchange and whether or not DNA synthesis
is involved in the recombination process. Most evidence now favors a model in
which homologous recombination is initiated by a double- strand break in one of
the DNA duplexes and the homologous, intact chromosome is used as a template to
repair the break by DNA synthesis.
Figure 12-31
.
Double-strand break model of meiotic recombination developed from
studies in the yeast S. cerevisiae
A pair of homologous chromatids (double-stranded DNA molecules) are
shown, one in blue and the other red. The darker and lighter shades
indicate complementary DNA strands. Alleles are indicated by capital
and lowercase letters (D, d). Complementary DNA
strands are also indicated by the presence or absence of prime signs
(for instance D and D′, and c and c′). In this
example, the initial double-strand break and resection of
5′ ends occurs on the a chromatid, removing
the d′ marker 1. This is
followed by strand invasion 2 and DNA synthesis with
the α chromatid D strand as the template
3. Repair synthesis of the other a
strand (using its complementary section on the α strand)
and ligation result in formation of a Holliday structure with two
crossovers 4a and 4b. (Repaired regions
are marked by black dashed lines.) Resolution of this crossed-strand
intermediate can occur in two ways. Cleavage at sites 2 and 4 (step
5b), or at sites 1 and 3 (not shown here), yields
nonrecombinant chromosomes, since all markers surrounding the
crossover site (i.e., to the left of c and to the
right of E) are derived from the same initial
chromosome. One duplex contains a complementary
D/D′ region, but the other contains a
heteroduplex mismatched d/D′ region
(yellow). In contrast, cleavage (step 5a) at sites 2
and 3, or at sites 1 and 4 (not shown here), yields recombinant
double-stranded DNA molecules, since all markers to the left and
right of the crossover site have undergone reciprocal recombination.
Note that one duplex contains a complementary
D/D′ region, but the other contains a
heteroduplex, mismatched d/D′ region
(yellow). Cells can repair such mismatched heteroduplex regions by
excising a single-strand segment containing the mismatch and using
the other strand as a template for synthesis of a matching strand
6 (see Figure
12-24). In this example, d is removed
and D is synthesized (jagged red segment), thus
“converting” d to
D. The opposite
D → d conversion
occurs with equal frequency.
If left unrepaired, double-strand breaks lead to broken
chromosomes and cell
death. The repair mechanism that joins the cut ends of nonhomologous
chromosomes
reconstitutes double-stranded DNA but deletes several
nucleotides (see
Figure 12-28). The mechanism outlined in
, however, completely
repairs a double-strand break, yielding a molecule of the same length as the
original. This model is thought to apply to homologous
recombination in
prokaryotes and during
meiosis in yeast and probably other
eukaryotes, as well
as to the error-free repair of double-strand breaks in DNA induced by radiation
and other agents.
As depicted in , a
double-strand break occurs in one of a pair of aligned homologous
double-stranded DNA molecules (the
a chromatid); the break is
enlarged to gaps by action of
5′ → 3′ exonucleases,
resulting in single-stranded 3′ ends (step 1). The 3′ end of
one
a strand then invades the homologous α
chromatid
(step 2) and is elongated at its 3′ end by
DNA polymerase, using the D
complementary strand of the α
chromatid as
template (step 3). Then the
3′ end of the other cut
a strand is elongated using the
other strand (D′) of the α
chromatid as
template, producing
an intermediate structure that contains two regions of
heteroduplex DNA and two
Holliday junctions (step 4). Following rotation at the crossover points, each
Holliday junction is resolved by cleavage of two single strands and ligation,
yielding two intact double-stranded DNA molecules (step 5a or 5b). Note that the
original double-strand cut in the
a chromatid has been fully
repaired.
This double-strand break model can account for the nonmendelian segregation of
certain markers that has been observed during meiotic recombination. This
phenomenon is most easily studied in yeast in which all four meiotic products
can be scored in the haploid progeny spores. In a cross of multiply-marked yeast
strains that undergo recombination, most allelic markers segregate according to
the Mendelian 2:2 ratio, but those located near the crossover point
exhibit 3:1 or 1:3 segregation. Such a nonreciprocal event
is called gene conversion because
one allele is apparently “converted” into another. Gene
conversion is thought to result from the process for resolving the
crossed-strand Holliday intermediate produced in the double-break model of
recombination.
To see how
gene conversion occurs, consider the example depicted in in which the
D (or
complementary D′) and
d markers lie between the two crossovers in the
intermediate. Of the two
recombinant DNA duplexes that result from cleavage and
ligation of the intermediate (i.e., the two products of step 5a), one contains
the
D marker in one strand and the
complementary
D′ in the other, whereas the other is a
heteroduplex with
D′ in one strand and the allelic
d in the other. Similarly, of the two nonrecombinant DNAs
that would result from cleavage of the intermediate at different sites (step
5b), one is also
heteroduplex in the
d/D segment. Mispairing of
the
D′ and
d alleles in the
heteroduplexes is recognized by the mismatch-repair system. Half of the time,
mismatch repair will convert the
D′
/d heteroduplex into
D′
/D (step 6); in this case the
d′
/d marker in the original
a chromatid is “converted” into
D′
/D. The other half of the
time, the
D′
/d heteroduplex will be
converted into
d′
/d, in which case
there is no
gene conversion. Thus three out of four
haploid yeast progeny
spores, each carrying one meiotic product, will show the
D
phenotype, that is, the 3:1
segregation ratio that typifies
gene conversion.
The Activities of E. coli Recombination Proteins Have Been
Determined
Three different but related enzymatic pathways carry out homologous
recombination
in
E. coli. All three pathways utilize the basic double-strand
break mechanism depicted in
to generate a Holliday-type structure, which undergoes branch migration followed
by endonuclease cleavage and then ligation to yield recombinants. In this
section, we describe the
enzymes that catalyze the primary
E.
coli recombination pathway and briefly consider homologous
eukaryotic
enzymes. These
enzymes were uncovered through genetic analysis of
recombination in
E. coli and subsequently purified.
Initiation of Recombination (RecBCD Enzyme)
The most common way that
E. coli cells generate a
recombinogenic single-stranded region of DNA, equivalent to step 1 in , probably is by action of
the RecBCD
enzyme, which has
helicase and exonuclease activities. This
enzyme complex, composed of
proteins encoded by the
recB,
C, and
D genes, specifically recognizes
double-strand breaks. Such breaks occur naturally during bacterial
conjugation, a process in which chromosomal DNA is transferred from one
bacterium to another through direct cell contact, and during
bacteriophage-mediated transduction. Double-strand breaks also can be
generated by exposure to x-rays and certain chemicals.
Figure 12-32
.
Initiation of recombination by E. coli
RecBCD enzyme
This multifunction enzyme, which binds to free blunt ends of DNA,
acts as a helicase,
3′ → 5′
exonuclease, and
5′ → 3′
exonuclease. When the enzyme encounters a CHI site, its
3′ → 5′
activity is inhibited and its
5′ → 3′
activity enhanced, yielding a single-stranded
3′-hydroxyl end. This recombinogenic 3′ end
becomes coated with multiple RecA proteins, which then catalyze
strand invasion and formation of a Holliday structure. [See D.
G. Anderson and S. C. Kowalczykowski, 1997,
Cell
90:77.]
The mechanism of action of RecBCD was worked out in studies with
bacteriophage λ. Certain regions of λ
phage DNA,
termed
CHI sites, undergo
recombination at higher
frequencies than other regions in normal
E. coli host cells
but not in
recBCD mutant host cells. Experiments with
purified RecBCD
enzyme and λ DNA indicate that the
protein
complex recognizes and binds to a free blunt end of the λ
phage
chromosome (equivalent to a double-strand break). The
enzyme then moves
along the DNA, its
helicase activity unwinding the duplex as it goes (). Initially, RecBCD
degrades both single strands one
nucleotide at a time using its dual
5′ → 3′ and
3′ → 5′ exonuclease activities.
However, when RecBCD encounters a CHI site, its 3′
→ 5′ exonuclease activity is inhibited and
its 5′ → 3′
exonuclease activity is enhanced. Thus, after passing a CHI site, RecBCD
begins to generate a single-stranded 3′-hydroxyl end. After the
resulting
recombinogenic end becomes coated with multiple
RecA
proteins, it can participate in the process of strand invasion.
Strand Invasion, Homologous Pairing, and Formation of Holliday-Type
Structure (RecA Protein)
Figure 12-33
.
Formation of Holliday-type structure by E.
coli RecA
In the presence of ATP, RecA binds single-stranded DNA (ssDNA)
and promotes insertion of the bound strand at a homologous
region of double-stranded DNA (dsDNA), yielding a crossed-strand
Holliday-type structure. The insertion reaction requires the
ATPase activity of RecA. [See S. S. Flory et al., 1984,
Cold Spring Harbor Symp. Quant. Biol.
49:513.]
Biochemical experiments showed that the
protein encoded by the
recA gene can bind to any single-stranded DNA (ssDNA).
In the presence of a homologous target duplex DNA, the RecA-ssDNA complex
can carry out two remarkable functions. First, RecA aligns the ssDNA with
its homologous target double-stranded DNA region and forms a complex with
it. Second, RecA inserts the ssDNA into the target DNA, displacing one of
the preexisting strands and forming a
heteroduplex Holliday-type structure
().
In subsequent studies, RecA was shown to bind to the single-stranded
recombinogenic ends generated by action of the RecBCD
enzyme. In fact, the
ability of RecA to load onto single-stranded DNA is stimulated by RecBCD
once it is activated by traversing a CHI site in the DNA (see ).
E.
coli SSB
protein stimulates this reaction by binding to the
single-stranded region and preventing intrastrand
base pairing, which would
inhibit binding of RecA. In the presence of ATP, RecA coats the
single-stranded region and polymerizes, forming a filament that wraps around
the entire length of the recombinogenic end. Because the polymerization of
RecA occurs in the
5′ → 3′ direction
along the DNA, coating takes place in a discontinuous fashion as a region of
the duplex is unwound. X-ray crystallographic analysis of RecA suggests that
each molecule has two DNA-binding sites, both of which may lie within the
core of the filament.
Branch Migration and Resolution of Holliday Structures (Ruv
Proteins)
Figure 12-34
.
Experimental demonstration of branch migration catalyzed by
E. coli RuvA and RuvB proteins
Complementary strands are indicated by darker and lighter shades
and the presence or absence of prime signs (A,
A′; B,
B′); segments with
different sequences are indicated by color. (a) A synthetic
Holliday structure was produced by annealing four synthetic
single-stranded oligonucleotides in which only the center
crossover region (green) was homologous and the opposite ends of
any given strand are, respectively, complementary to two
different strands. (b, c) Treatment of the Holliday structure
with RuvA and RuvB in the presence of ATP leads to branch
migration followed by unwinding to yield cruciform structures
with nonhomologous single-stranded ends. Branch migration in the
other direction (towards A and
D) yields similar cruciform structures.
[Adapted from H. Iwasaki et al., 1992, Genes &
Dev.
6:2214.]
Although formation of
Holliday structures depends on RecA, their maintenance
does not. Removal of RecA leaves stable
Holliday structures, which have been
used as
substrates in studies on branch migration and
resolution. Migration
of the crossover point, which can be detected in synthetic Holliday
structures, is efficiently catalyzed by
E. coli RuvA and
RuvB
proteins ().
Further analysis showed that RuvA specifically recognizes the Holliday
junction, whereas RuvB has the
helicase activity necessary for promoting the
observed branch migration.
Figure 12-35
.
Action of E. coli proteins in branch
migration and resolution of Holliday junctions
(a) Model of the association of RuvA and RuvB with a Holliday
junction as determined by electron microscopy. For clarity the
proteins are drawn behind the DNA; in reality the DNA passes
through the central hole of each RuvB hexamer. Powered by ATP
hydrolysis, the ringlike RuvB molecules rotate the DNA double
helices inside them, much in the way a screw is rotated inside a
nut. The two RuvB
proteins impart equal and opposite rotational
forces to the DNA, as indicated by the black circular arrows.
The straight arrows indicate the direction of movement of DNA
through the RuvA/RuvB complex. (b) Diagram illustrating
migration and
resolution of a Holliday junction. (
Alleles and
complementary strands are indicated as in .) Movement of a junction
through the RuvA/RuvB complex generates
heteroduplex regions,
exactly as observed in the experiment shown in . Following
branch migration
1, two molecules of
the RuvC endonuclease bind to RuvA
2 and cut the intermediate at two
points that are 180° apart
3 . Ligation of the cut ends
completes
resolution
4 . In this
example, two recombinant molecules are formed, but if RuvC
cleaved at the two other points, nonrecombinant DNAs would be
produced (see ). [Adapted from S. C. West, 1996,
Cell
86:177; J. Raftery et al., 1996,
Science
274:415; and A. Kuzminov, 1996,
BioEssays
18:757.]
Recent studies have clarified the action of these two
proteins. The active
tetrameric form of RuvA binds to the center of a Holliday junction,
unfolding the junction into a square planar configuration and keeping the
four single-stranded segments apart. This induces binding of two ringlike
hexameric RuvB
proteins, which surround the double-stranded DNA exiting from
opposite sides of the RuvA complex (). Powered by ATP
hydrolysis, the RuvB rings act as
molecular
pumps, pulling two double-stranded DNAs into the RuvA complex,
separating the strands, and then extruding two double-stranded
heteroduplexes out of the RuvA complex. Following branch migration, two RuvC
endonuclease
proteins bind to the RuvA/ RuvB complex and then cut the DNA
intermediate at two sites 180° apart; subsequent ligation generates
recombinant (or nonrecombinant) molecules containing a segment of
heteroduplex DNA ().
Homologous Eukaryotic Recombination Enzymes
Although RecA, RecBCD, and RuvA, B, and C were initially identified in
E. coli, all eukaryotic cells, including human cells,
produce proteins of similar structure and function. For instance, the human
and yeast RAD51 proteins, which are homologous in sequence, catalyze pairing
of homologous DNA segments and DNA strand insertion similarly to RecA. A
Topo II – like protein encoded by the yeast
Spo11 gene generates the double-strand breaks that
occur during meiotic recombination, and homologous proteins are found in
bacterial and other eukaryotic cells. Thus the molecular mechanism of
homologous recombination most likely is similar in all types of cells.
Cre Protein and Other Recombinases Catalyze Site-Specific
Recombination
Homologous recombination occurs randomly between two homologous DNA segments, and
there is relatively little specificity as to the site at which the actual
crossover occurs. In site-specific recombination, a different
type of process, relatively short, unique nucleotide sequences in two DNA
molecules are recognized by enzymes called recombinases, which
then catalyze the joining of the two molecules. Several examples of
site-specific recombination have been discovered in both prokaryotic and
eukaryotic cells.
One well-studied example, the integration of bacteriophage λ into a
particular site in the E. coli chromosome (see Figure 6-19), is catalyzed by a viral
enzyme called integrase. The genome of λ phage
contains a 15-bp attachment site whose 7-bp core sequence is identical with the
integration (or attachment) site in the host-cell DNA. Integration can be
carried out in a cell-free reaction system with only two purified proteins,
λ integrase and integration host factor, a cellular protein.
Integrase also catalyzes the reverse reaction, excision of the circular
λ phage DNA from a bacterial chromosome.
Figure 12-36
.
Cre protein, encoded by phage P1, catalyzes site-specific
recombination between loxP sites on multimeric circles of P1 DNA,
generating a circular monomeric DNA
Repetition of this process eventually converts the entire multimeric
DNA into circular P1 monomers.
The site-specific
recombination reaction that is best understood at the molecular
level is catalyzed by Cre, a
protein encoded in the
genome of bacteriophage P1.
During
phage P1 DNA replication, long
multimeric DNAs are produced; these are
resolved into
monomeric P1 DNAs by
recombination at loxP sites, which separate
the P1 DNA
monomers composing a
multimeric DNA (). The Cre
protein that catalyzes this reaction is
similar in structure and function to λ integrase. Moreover, the
excision of
monomeric P1 DNAs from a
multimeric circle by Cre is mechanistically
similar to the excision of λ DNA from the bacterial
chromosome by
integrase.
Figure 12-37
.
Mechanism of Cre-loxP site-specific recombination
Two Cre proteins bind on either side of each of two loxP sites, and
then associate together, forming a synapse composed of two DNA
molecules and four Cre proteins
1 . A tyrosine (Tyr) residue on
opposite Cre proteins cleaves the DNA strand to which the proteins
are bound, forming two covalent 3′ phosphotyrosine bonds
and two free 5′-hydroxyl ends
2 . The free
5′-hydroxyl ends in this Cre-DNA intermediate then react
with the opposite phosphotyrosine bonds, yielding a Holliday-type
intermediate and regenerating native Cre proteins
3 . Next, a tyrosine residue on the other
two Cre proteins at the synapse cleave the DNA strands to which they
are bound, again forming covalent 3′ phosphotyrosine bonds
and free 5′-hydroxyl ends to give a second Cre-DNA
intermediate 4 . Finally, the
free 5′-hydroxyl ends of this intermediate attack the
opposite phosphotyrosine bonds, yielding the recombinant DNA
products still bound to regenerated native Cre proteins
5 . [Adapted from F. Guo, D.
Gopaul, and G. Van Duyne, 1997, Nature
389:40.]
Figure 12-38
.
Ribbon model based on x-ray crystallography of the Cre-DNA
intermediate II in site-specific recombination (see )
Each of the cleaving subunits is covalently linked to a DNA strand by
a phosphotyrosine bond. [Courtesy of Dr. G. Van Duyne.]
The mechanism of Cre-catalyzed
recombination is depicted in . This mechanism involves the sequential
formation of two transient intermediates in which Cre and DNA are covalently
linked by phosphotyrosine bonds, similar to those between DNA and
topoisomerase
II (see
Figure 12-14,
inset). Reaction of the first Cre-DNA intermediate
generates a Holliday-type structure; reaction of the second one yields the
recombinant double-stranded DNA products. By using DNA molecules with
mutations
or single-strand breaks in the short loxP homologous recognition sites,
researchers have been able to stop the Cre-catalyzed reaction at several stages
and collect intermediates ().
The P1 recombinase system has proved useful to mouse geneticists. Because many
genes are required at multiple stages of development,
“classical” gene-knockout mice, produced by the procedure
depicted in Figure 8-34, frequently die
as early embryos. However, the P1 loxP-Cre recombinase system has enabled
geneticists to generate animals in which a particular gene of interest is
deleted only in one specific tissue (see Figure
8-35). Such tissue-specific knockout mice enable researchers to study
the function of any gene in just one tissue of the adult animal. Thus, an
understanding of how one site-specific recombination system excises a DNA
segment out of a specific chromosomal site led to the development of an
important tool for mouse geneticists.
SUMMARY
-
During homologous recombination, two duplex
DNA molecules are broken and strands are exchanged. This process, which
occurs randomly along the genomes of all organisms, plays an important
role in generating genetic diversity.
-
Double-strand breaks in DNA initiate most
cases of homologous recombination. The break becomes enlarged to gaps,
forming single-stranded 3′ recombinogenic ends that invade the
other duplex. Repair synthesis of the missing regions forms an
intermediate containing two crossed-strand Holliday junctions.
Resolution of this intermediate occurs by rotation followed by cleavage
and ligation of two strands at each Holliday junction (see ). -
In E. coli, a
recombinogenic end created by the RecBCD enzyme complex is stabilized by
binding of RecA protein (see ). Catalyzed by RecA, the single-stranded 3′
end then pairs with and invades a homologous duplex DNA segment, forming
an intermediate containing two regions of heteroduplex DNA and two
Holliday junctions. After branch migration, catalyzed by RuvA and RuvB,
two strands at each junction are cut by RuvC, an endonuclease, and then
ligated to yield two duplex DNA molecules (see ). -
Because eukaryotic cells express proteins
homologous to the E. coli recombination proteins, all
cells are thought to carry out homologous recombination by a similar
molecular mechanism.
-
Site-specific recombinases recognize,
cleave, and recombine short homologous DNA sequences in two different
DNA molecules.
-
During site-specific recombination
catalyzed by phage P1 Cre, transient phosphotyrosine bonds are formed
between bound Cre molecules and the cut 3′-hydroxyl ends of
the DNA duplexes (see ). Integration of phage λ, catalyzed by
integrase, is thought to occur by a similar mechanism.
ǀ
