The Nucleus and Gene Expression
Making crossovers during meiosis
M.C. Whitby1
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
Abstract
Homologous recombination (HR) is required to promote both correct chromosome segregation and genetic
variation during meiosis. For this to be successful recombination intermediates must be resolved to generate
reciprocal exchanges or ‘crossovers’ between the homologous chromosomes (homologues) during the
first meiotic division. Crossover recombination promotes faithful chromosome segregation by establishing
connections (chiasmata) between the homologues, which help guide their proper bipolar alignment on
the meiotic spindle. Recent studies of meiotic recombination in both the budding and fission yeasts have
established that there are at least two pathways for generating crossovers. One pathway involves the
resolution of fully ligated four-way DNA junctions [HJs (Holliday junctions)] by an as yet unidentified
endonuclease. The second pathway appears to involve the cleavage of the precursors of ligated HJs, namely
displacement (D) loops and unligated/nicked HJs, by the Mus81-Eme1/Mms4 endonuclease.
The generation of gametes depends on a specialized form of
cell division called meiosis, which reduces cellular chromosome content from diploid to haploid. This involves two
consecutive cell divisions (called meiosis I and II) following
a single round of DNA replication (Figure 1). Segregation of
the homologous chromosomes (homologues) occurs during
meiosis I, whereas sister chromatids segregate during meiosis II. In many ways, meiosis II resembles a normal mitotic
cell division where cohesion between the sister chromatids is
used to help guide their proper segregation. However, proper
homologue segregation during meiosis I demands additional
layers of complexity. Part of this complexity involves the
stimulation of HR (homologous recombination) between
the homologues. The function of this recombination in most
organisms is threefold: (i) it promotes genetic diversity by
creating new and potentially beneficial combinations of
maternal and paternal alleles; (ii) it removes deleterious mutations that would otherwise result in the gradual decay
of genetic information [1]; and (iii) it establishes physical
connections (called chiasmata) between the homologues that
are important for their proper bipolar attachment to the
meiosis I spindle (Figure 1) (for a review, see [2]). Defects in
this latter function can result in aneuploidy through homologue missegregation.
Much of our current understanding of meiotic recombination has come from studies of meiosis in fungi. Here, the
four haploid products of a single meiosis can be analysed
genetically, and studies that have employed this tractability
have played a central role in the development of models for
HR. Foremost among these is the DSB (double-strand break)
repair model (Figure 2) [3]. This proposes that recombiKey words: chiasma, crossover, Holliday junction, homologous recombination (HR), meiosis,
Mus81.
Abbreviations used: D-loop, displacement loop; HJ, Holliday junction; dHJ, double HJ; DSB,
double-strand break; HR, homologous recombination; MMR, mismatch repair; SDSA, synthesisdependent strand annealing; SEI, single end invasion.
1
email [email protected]
nation is initiated by the formation of a DSB in one of
the homologues. The broken DNA ends are then resected
by degradation of their 5 -ended strands to generate singlestranded tails with 3 -OH termini (Figure 2, step 2). One of
these tails then locates its equivalent sequence on the homologue and invades it to form a displacement loop (D-loop)
[this is also referred to as an SEI (single end invasion)] (Figure 2, step 3). DNA synthesis primed by the invading strand
extends the D-loop, enabling it to anneal to the singlestranded tail on the other side of the break (second end
capture) (Figure 2, steps 4–5). Further DNA synthesis together with the ligation of strand breaks results in the homologues being connected by two four-way DNA junctions – a
structure called the dHJ (double Holliday junction) (Figure 2,
step 6). The dHJ can be resolved by cleavage of a pair of
symmetrical strands at each junction. Cleavage of a different
pair of strands at each junction splices together maternal
DNA flanking one side of the dHJ with paternal DNA
flanking the other side (Figure 2, steps 6 and 7). This type of
recombinant product is called a crossover. It constitutes a new
combination of alleles and establishes a chiasma. However, if
the dHJ is resolved by cleavage of the same pair of strands at
each junction, then crossing over does not occur (Figure 2,
steps 6 and 7). Such non-crossover recombinants do not
contribute to chiasma formation.
Since its proposal in 1983, many aspects of the DSB repair model have been substantiated by physical detection of
intermediates, primarily in the budding yeast Saccharomyces
cerevisiae. These include the DSBs, resected ends, SEIs and
dHJs (see e.g. [4–8]). Many of the enzymes that catalyse its
various stages have also been identified, and found to be
evolutionarily conserved. These include a complex of proteins
involving Spo11 that makes DSBs, the Mre11–Rad50–Xrs2
complex that is involved in strand resection, and Rad51,
which, together with Dmc1 and various accessory proteins,
drives the central reactions of homologous pairing and strand
invasion that lead to the formation of the dHJ (for reviews,
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Figure 1 Meiotic chromosome segregation
Recombination during meiosis I establishes connections (chiasmata) between the homologues that are mediated by sister
chromatid cohesion. These connections help to guide the proper monopolar attachment of the homologues to the meiosis I
spindle. Once all the chromosomes are correctly attached, cohesin along the chromosome arms is degraded, and the
homologues are divided. Cohesion is maintained at the centromeres during the meiosis I division in order to guide correct
bipolar attachment of the sister chromatids to the meiosis II spindle.
Figure 2 The Szostak et al. [3] DSB repair model
Dashed lines represent newly synthesized DNA. Note that noncrossovers are also formed by cleavage at A + B, and crossovers by
cleavage at A + b. See main text for further details.
see [9,10]). However, the identity of the nuclease that cleaves
the dHJ has remained elusive. In some ways, this is surprising
because HJ resolvases that are involved in recombination
reactions in bacteriophage, eubacteria, archaea, eukaryotic
viruses and yeast mitochondria have been identified and
characterized extensively [11].
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The DSB repair model predicts that both crossover and
non-crossover recombinants derive from dHJ resolution [3].
This is consistent with the activities of known HJ resolvases
that would cleave either pair of strands at each HJ with equal
probability. However, in recent years, a number of mutations
have been found in S. cerevisiae that reduce dHJ resolution
and crossover formation without affecting the formation of
DSBs and non-crossovers (reviewed in [12]). This indicates
that crossovers and non-crossovers derive from independent
pathways of DSB repair. In fact, it appears that dHJs are
resolved exclusively as crossovers (Figure 3, steps 6a and 7a),
whereas non-crossovers may stem from a mechanism called
SDSA (synthesis-dependent strand annealing), in which
dissociation of the SEI after DNA synthesis is followed by
annealing of the two broken ends (Figure 3, steps 3b–6b)
[13,14].
How does the cell direct some DSBs to be repaired by
the crossover pathway and others by the non-crossover
pathway? At least a part of the answer involves the recruitment of a set of meiosis-specific proteins (Zip1, Zip2, Zip3,
Mer3, Msh4 and Msh5) to certain DSB sites [13,15]. These
proteins, often referred to as the ZMM proteins, seem to
promote SEI stability and dHJ formation, and ensure that
resolution occurs with the appropriate bias to generate crossovers. Without them, SEIs appear to be transient and unstable
structures, which cannot be detected by current physical
analyses [13]. It is assumed that these unstable SEIs are
channelled down alternative pathways such as SDSA [12].
Recent biochemical studies have shown how three of the
ZMM proteins (Mer3, Msh4 and Msh5) could promote
the stability of early strand invasion intermediates. Mer3 is
a DNA helicase, which in vitro can extend the region of
heteroduplex DNA formed during strand invasion catalysed
by Rad51 (Figure 3, steps 3a and 4a) [16]. It is easy to see
how this activity would promote SEI stability by increasing
the number of base pairs linking the two homologues. Msh4
and Msh5 are homologues of the bacterial MMR (mismatch
repair) protein MutS, which binds to mismatched DNA
via one of two central holes in its homodimeric structure
The Nucleus and Gene Expression
Figure 3 Current models for meiotic DSB repair
See main text for details.
[17]. Msh4 and Msh5 are not involved in MMR, and recent
studies of the human homologues of these enzymes (hMSH4
and hMSH5) have shown that, instead of binding to mismatched DNA, they form a heterodimeric complex that binds
specifically to HJs, including the half-HJs that are formed
upon strand invasion (Figure 3, step 3a) [18]. The hMSH4–
hMSH5 complex appears to encircle the HJ, with possibly
each of its two central holes binding a different DNA duplex.
Upon binding ATP, the complex releases from the HJ and
slides along the adjacent duplex DNA. It is thought that
this would allow further hMSH4–hMSH5 heterodimers to
bind to the HJ (Figure 3, step 4a) [18]. The accumulation of
these complexes encircling the recombination intermediate
could help to stabilize it and direct the manner in which it is
resolved, ensuring that only crossovers are formed (Figure 3,
steps 5a–7a) [18,19].
During MMR in Escherichia coli, MutS interaction with
MutL activates the latent endonuclease activity of MutH
[17]. MutH then nicks a hemimethylated GATC site located
within approx. 1 kb of the mismatch, and this allows entry of
a DNA helicase and exonuclease to remove a stretch of singlestranded DNA containing the mismatch. In S. cerevisiae,
homologues of MutL called Mlh1 and Mlh3 form a complex
that functions downstream of Msh4–Msh5 to promote crossover formation [20–24]. This suggests that there is an endonuclease that is activated by the interaction of Msh4–Msh5
with Mlh1–Mlh3, which resolves dHJs. However, a homologue of MutH has not been found in eukaryotes and
the identity of an endonuclease that might be controlled
and/or activated by Msh4–Msh5 and Mlh1–Mlh3 remains
elusive. An HJ resolvase activity, which depends on two
Rad51-like proteins, Xrcc3 and Rad51C, has been detected
in fractionated mammalian cell extracts [25]. Budding yeast
does not contain direct homologues of Xrcc3 and Rad51C so
it is unclear whether it uses a related resolvase for crossover
formation. However, the plant Arabidopsis thaliana does
contain these proteins, and moreover they seem to be critical
for a late stage in meiotic recombination [26,27]. Like budding
yeast, Arabidopsis has a crossover pathway that depends on
MSH4 and MER3, but it is not known whether Xrcc3 and
Rad51C are part of this pathway [28,29].
In S. cerevisiae, the majority of DSBs are repaired as noncrossovers, whilst comparatively few (∼90 in the entire
genome) are repaired as crossovers. The distribution of
these crossovers is non-random with both large and small
chromosomes receiving at least one crossover and with crossovers tending not to be near one another on the same chromosome. This phenomenon of crossover interference ensures
that crossovers are evenly spaced and depends on the
recruitment of the ZMM proteins to particular DSB sites at
an early stage in their repair [12,13,15]. The ZMM-dependent
pathway is therefore synonymous with what is often referred
to as the crossover interference pathway. It is not clear
what underlies crossover interference (i.e. prevents ZMM
proteins loading at neighbouring DSB sites). One simple idea
is that consecutive loading of Msh4–Msh5 at a recombination
intermediate at one site results in a localized depletion of these
enzymes, preventing their loading at neighbouring sites [19].
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Other models invoke concepts whereby the commitment
of one site to become a crossover sends out a ‘signal’ to
neighbouring sites, which inhibits crossover formation (discussed in [15]). Such a signal could involve the imposition and
relief of stress being transmitted along each homologue axis
[30].
The dependence on the ZMM proteins for crossover
formation is not absolute in budding yeast. In fact, at 30◦ C,
ZMM mutants typically retain approx. 50% of the normal
level of crossovers, although, at 33◦ C, this is reduced to
15% [13]. Most of these non-ZMM crossovers depend on
the Mus81 and Mms4 proteins (the homologue of Mms4
in fission yeast and humans is called Eme1) [24,31]. These
function in a distinct pathway of crossover formation that is
not subject to crossover interference. In a few organisms, such
as the fission yeast Schizosaccharomyces pombe, crossover
formation depends almost entirely on this Mus81-dependent
pathway [32,33]. This correlates with the absence of ZMM
proteins and the lack of crossover interference in these
organisms [34].
Mus81 is related to the XPF (xeroderma pigmentation
group F) family of structure-specific endonucleases and it
forms a complex with Mms4/Eme1 that can cleave a variety of branched DNA structures in vitro, including HJs
(reviewed in [35]). In fact, initial biochemical and genetic
analysis of Mus81–Eme1 in fission yeast had indicated that
it was the main HJ resolvase in this organism [36]. The key
observations here were that it could cleave HJs in vitro and
that mutation of mus81 dramatically reduced spore viability
(an indicator of meiotic failure), which could be rescued by
the heterologous expression of a bacterial HJ resolvase [36].
However, extensive analysis of the substrate specificity of
Mus81–Eme1/Mms4 from both budding and fission yeast, as
well as from humans, has shown that this enzyme is actually
far more active on other kinds of branched DNA structures
than it is on intact HJs [37–41]. In particular, it favours
D-loops and nicked HJs [32,42]. This substrate specificity
suggests that, instead of resolving dHJs, Mus81–Eme1/Mms4
might act earlier in the recombination reaction to cleave the
D-loop that is formed by the SEI and the unligated HJ that is
formed by second end capture (Figure 3, steps 3c and 5c) [32].
An appealing aspect of this model is that the way in which
Mus81–Eme1/Mms4 cleaves each of these junctions would
produce an obligate crossover in vivo (Figure 3, step 6c).
This is consistent with genetic data showing that mutation
of mus81 only affects crossover formation [32,33]. It also
provides a simple explanation for why the overriding majority
of DSBs in Schiz. pombe, unlike in S. cerevisiae, are repaired
as crossovers [43,44].
In budding yeast, it is thought that the Mus81-dependent
pathway is largely a backup for the ZMM-dependent pathway of crossover formation. Presumably, the loading of ZMM
proteins at early recombination intermediates shields them
from Mus81–Mms4 and allows the dHJ to form. Another
MMR protein, Mlh2, may also be important here for preventing Mus81 from accessing the DNA [45]. Utilization of
a ZMM or equivalent pathway, which is subject to crossover
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interference, appears to be the principal pathway of crossover
formation in most organisms [34]. Like budding yeast, some
organisms such as Arabidopsis may also employ a Mus81dependent pathway for a proportion of their crossovers
[28,29]. However, others, such as the worm Caenorhabditis
elegans, appear to depend solely on a ZMM or ZMM-like
pathway [34]. Recently, a MUS81 knockout mouse was made
and found to be perfectly fertile and normal for gametogenesis
[46]. In contrast, msh4−/− and msh5−/− mice are sterile and
exhibit defects in chromosome pairing during meiosis I
[47–49]. Likewise, both mlh1−/− and mlh3−/− mice are sterile
and exhibit severe defects in crossing over [50,51]. These
results suggest that mammals, like worms, may employ only
the ‘ZMM’ type of pathway for crossover formation.
Are there other pathways of crossover formation? In
budding yeast, a few crossovers are formed in an mlh1−
mms4− double mutant, and this number actually increases by
approx. 3-fold when MSH5 is also deleted [24]. These results
suggest that there is a third pathway that is normally inhibited
by Msh5. However, this pathway, rather than promoting the
production of viable meiotic products (spores), is detrimental
to it, since spore viability is much higher in the mlh1− mms4−
double mutant than in the mlh1− mms4− msh5− triple mutant [24]. Possibly, this ‘third pathway’ is simply a product of
the elusive HJ resolvase from the ZMM-dependent pathway
being left to function without its normal activators and
controllers. Although it might resolve some dHJs successfully, its being unfettered may allow it to cleave DNA that it
is not supposed to, resulting in loss of viability.
The last few years have seen many exciting developments
in the recombination field, which have given us a much
clearer picture of how crossovers are formed during meiosis.
However, before we can really get to grips with understanding
the mechanics of crossing over, the elusive HJ resolvase
from the ZMM pathway must be identified. Concerted efforts
are being made, so hopefully we will not have to wait long.
I thank Fekret Osman for comments on this paper. Work in my
laboratory is funded by a Wellcome Trust Senior Research Fellowship
in Basic Biomedical Research.
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Received 20 June 2005
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