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INTRODUCTION |
DNA replication complexes (replisomes) routinely encounter
blockages in the DNA that they are copying (1). Obstructions include
transcription complexes, DNA repair complexes and bulky DNA lesions
(e.g. pyrimidine dimers). It is imagined that the cellular
response to each type of blockage differs according to parameters that
have yet to be clearly defined. In principle if the blockade is another
protein complex it would make sense for the replisome to suspend
synthesis until the obstruction has moved out the way. However, simply
waiting for a blocking lesion to be removed may not be an option for
the replisome since it may itself be blocking repair complexes from
accessing the damaged DNA. In such circumstances polymerases that are
able to function on damaged templates can be recruited (2). In
Escherichia coli these polymerases are error-prone and
therefore appear to be used only when there is an excess of DNA damage
(3). However, in eukaryotes some of these polymerases are not
inherently mutagenic and therefore they may be used more readily (2).
If the lesion is in the lagging template strand then synthesis of the
relevant Okazaki fragment may be skipped resulting in a
lesion-containing DNA gap that is dealt with by post-replication repair
(4). Alternatively lesions in either the leading or lagging template strand may provoke the partial or complete disassembly of the replisome
followed by the spontaneous or enzyme-driven reversal of the
replication fork (5-9). Fork reversal (also referred to as fork
regression) involves the unwinding of nascent strands and re-pairing of
parental strands. This may serve both to provide room for lesion repair
and/or a means of unfettering nascent strands to act in a template
switching mechanism for the bypass of the lesion (10). Depending on the
extent of fork reversal either one or both nascent strands are extruded
from the fork. In the latter case this will result in the formation of
a Holliday junction when the strands anneal. In order for replication
to restart reversed forks have to be reset. This can be achieved either
by the removal of the extruded DNA by a nuclease or reverse branch
migration of the junction point by a DNA helicase (6, 11-14).
Alternatively the reversed fork can be cleaved to detach either the
leading or lagging strand arm of the fork (6, 15). The exposed DNA end
is then targeted by recombination enzymes that both repair the break
and facilitate the reassembly of the replisome (16, 17).
In the fission yeast Schizosaccharomyces pombe the Rqh1 DNA
helicase and Mus81-Eme1 endonuclease have both been implicated in
processing DNA junctions at stalled replication forks (11, 18, 19).
Rqh1 is a member of the RecQ family of DNA helicases that includes Sgs1
in Saccharomyces cerevisiae and the Bloom's (BLM),
Werner's (WRN), and Rothmund-Thomson's Syndrome helicases in humans
(20). At least some RecQ helicases, including Rqh1, appear to limit
recombination at stalled replication forks (11, 21, 22). Both BLM and
WRN can bind and branch migrate Holliday junctions in vitro
prompting the suggestion that they could catalyze fork resetting and
thereby limit recombination caused by fork cleavage (12, 14). A similar
function has been ascribed to Rqh1. This is based on the observation
that an rqh1
mutant's chromosome segregation
defects, which are induced by replication fork stalling, are partially
suppressed by the E. coli Holliday junction resolvase RusA
(11). It would appear that in the absence of Rqh1 at least some
Holliday junctions at stalled replication forks remain unprocessed and
so impede sister chromatid segregation.
Mus81-Eme1 is related to the XPF-ERCC1 and Rad1-Rad10 family of
heteromeric structure-specific endonucleases that function in
nucleotide excision repair, DNA interstrand cross-link repair and
recombination (23, 24). mus81
and
eme1
mutants are hypersensitive to ultraviolet
light, the ribonucleotide reductase inhibitor hydroxyurea and the
alkylating agent methylmethane sulfonate that cause replication fork
stalling, and are inviable in the absence of rqh1 (18, 19,
24). These phenotypes are suppressed by RusA suggesting that Mus81-Eme1
cleaves Holliday junctions, and thereby provides an alternative to Rqh1
for processing junctions formed at stalled replication forks (18).
Indeed both S. pombe and human Mus81, purified from their
endogenous cells, cleave Holliday junctions in vitro (19,
25). However, when purified from E. coli cells, Mus81-Eme1
and its homologue from S. cerevisiae Mus81-Mms4 exhibit only
low levels of Holliday junction cleavage activity, whereas they cleave
fork substrates very well (18, 26). A strong preference for fork
substrates is also observed for Mus81 from HeLa cells (27). These
observations have prompted the suggestion that Mus81-Eme1 and
Mus81-Mms4 may cleave stalled replication forks before they have fully
reversed to form a Holliday junction (18, 26, 27).
To gain a better understanding of Mus81-Eme1's potential for
processing DNA junctions at stalled replication forks, we have tested
its ability to cleave a variety of fork substrates made from
oligonucleotides and
DNA molecules. Cleavage sites have also been
mapped and a direct comparison with Mus81-Mms4 made. From this study we
show that both Mus81-Eme1 and Mus81-Mms4 cleave structures that are
expected to form when a replication fork reverses. Cleavage always
occurs between 3 and 6 bp 5' to the junction point and in most cases is
targeted to the equivalent of the leading strand template. The one
exception to this rule is a substrate that mimics a replication fork
that has reversed to expose only its nascent lagging strand, which is
cleaved in the equivalent of the lagging strand template. The potential
significance of these data for fork processing in vivo is discussed.
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EXPERIMENTAL PROCEDURES |
Proteins--
Mus81-Eme1 and NLS-RusA-GFP were purified as
described previously (18). S. cerevisiae Mus81-Mms4 was
purified as described below. Amounts of protein were estimated by a
modified Bradford assay using a Bio-Rad protein assay kit and bovine
serum albumin as the standard. The concentration of NLS-RusA-GFP is
expressed as moles of monomers. Mus81-Eme1 and Mus81-Mms4 are assumed
to be heterodimers and therefore their concentrations represent moles of heterodimers.
Plasmids--
S. cerevisiae MUS81 and
MMS4 were amplified by PCR from a genomic DNA template using
primers that incorporate suitable restriction sites 5' and 3' of their
open reading frames to facilitate cloning. MUS81 was cloned
as an NdeI-BamHI fragment into pT7-7 to make pMW566, and MMS4 was cloned as an
NdeI-EcoRI fragment into pET14b (Novagen) to make
pMW567. A fragment containing the T7 phage Ø10 promoter and
MUS81 gene was then amplified by PCR from pMW566 using
primers that incorporate terminal SphI sites, and cloned into a unique SphI site upstream of the T7 phage Ø10
promoter in pMW567 to make pMW571.
Overexpression and Purification of Mus81-Mms4--
100 ml of
E. coli BL21-RIL cells (Stratagene) containing pMW571 were
grown with aeration at 25 °C in Luria-Bertani broth containing 125 µg/ml carbenicillin and 50 µg/ml chloramphenicol. At a cell density
corresponding to an OD600 of 0.5 Mms4, containing an
N-terminal His6 tag, and Mus81 were induced by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM. After a 2.5 h induction the cells were harvested by centrifugation, resuspended in 10 ml of Buffer
H (50 mM potassium phosphate, pH 8.0, 0.3 M
NaCl, 10% glycerol) and frozen at
80 °C until required. Frozen
cells were thawed at room temperature and then mixed on ice with
protease inhibitors, 10 mM
-mercaptoethanol, and 1%
Triton X-100 before lysis by passage through a French pressure cell at
30,000 p.s.i. After centrifugation at 43,700 × g for
50 min the supernatant was loaded directly onto a 1-ml
Ni-nitrilotriacetic acid superflow column (Qiagen), which was washed
with 30 ml of Buffer H + 50 mM imidazole before eluting bound Mus81-Mms4 with 2 ml of Buffer H + 200 mM imidazole.
Mus81-Mms4 was then stored as aliquots at
80 °C.
DNA Substrates--
Oligonucleotides 2, 5-8, 10-11, and 14 used to make the fork substrates have been described previously (18,
28). The sequence of oligonucleotide 15 is
5'-TGCCGAATTCTACCAGTGCCAGTGAT-3'. Oligonucleotides were supplied by
Sigma-Genosys Ltd. and were purified by electrophoresis through a 15%
(w/v) polyacrylamide gel containing 7 M urea, full-length bands being cut out and extracted from the gel by soaking in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) overnight.
F1-F6 were made by annealing the appropriate combination of
oligonucleotides as follows: F1, oligonucleotides 2, 5 11, and 14; F2,
oligonucleotides 2, 8, 10, and 15; F3, oligonucleotides 2, 5, 7, and
14; F4, oligonucleotides 2, 5, 6, and 11; F5, oligonucleotides 2, 5 and
11; and F6, oligonucleotides 2, 5 and 14. The procedures for annealing
and substrate preparation have been described previously (28, 29). DNA
substrates were labeled at the 5'-end of one of their component
oligonucleotides as indicated using [
-32P]ATP and
polynucleotide kinase.
,
Kpn, and
Sma
substrates were made as described by McGlynn and Lloyd (8).
Enzyme Assays--
,
Kpn, and
Sma cleavage reactions (10 µl) contained DNA and
enzyme as indicated in reaction buffer (25 mM Tris-HCl, pH
8.0, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin,
6% glycerol, 10 mM MgCl2). Reactions were
incubated at 30 °C for 30 min. They were stopped by the addition of
one-fifth volume of stop mixture (2.5% SDS, 200 mM EDTA,
10 mg/ml proteinase K) followed by a further 15 min at 30 °C to
deproteinize the mixture. Reaction products were then analyzed by
electrophoresis through a 0.8% agarose gel in Tris borate/EDTA (TBE)
buffer at 100 V for 80 min.
The cleavage reactions (30 µl) in Figs. 3 and 5 contained 10 nM of either Mus81-Eme1 or Mus81-Mms4 as indicated, and
between 0.4 and 3.6 nM of junction DNA (depending on the
oligonucleotide that is labeled), in the same reaction buffer as above.
Reactions were incubated and stopped as above, and the products
analyzed by electrophoresis through 10% native polyacrylamide gels in
TBE buffer at 200 V for 2 h and 15% denaturing gels containing 7 M urea. For native gels, deproteinated reactions
were mixed with loading dye and loaded directly onto the gel. For
denaturing gels, reactions were extracted with
phenol/chloroform/isoamyl alcohol (25:24:1), and the DNA was
precipitated with ethanol, resuspended in gel-loading buffer (0.05%
(w/v) bromphenol blue, 0.05% (w/v) xylene cyanol, 10 mM
EDTA, pH 7.5, 97.5% (v/v) formamide), and denatured by boiling for 2 min before loading onto the gel. To map cleavage sites reaction
products were run alongside Maxam-Gilbert sequence ladders of the
appropriate labeled oligonucleotide. A 1.5-base allowance was made to
compensate for the nucleoside eliminated in the sequencing reaction.
The cleavage reactions (20 µl) in Fig. 4A contained the
indicated amount of Mus81-Eme1 and 0.3 nM of junction DNA
(labeled in oligonucleotide 5) in reaction buffer. Reactions were
incubated, stopped, and then analyzed by electrophoresis through a
native polyacrylamide gel as described above. For measurement of the rates of cleavage shown in Fig. 4B, 60-µl reactions were
set-up as above, and 10-µl aliquots withdrawn and stopped at the
indicated times. The data shown are the means of two independent
experiments. All gels were dried onto 3 mm Whatman paper and analyzed
by phosphorimaging using a Fuji FLA3000.
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RESULTS |
Mus81-Eme1 Cleaves
DNA Poorly--
Previously we have shown
that Mus81-Eme1 cleaves small synthetic Holliday junctions (X-12)
relatively poorly compared with fork substrates (18). X-12 contains
only a 12-bp homologous core in which the junction crossover point
branch migrates, and therefore could in theory be cleaved inefficiently
by Holliday junction resolvases that are affected by sequence context.
To see if Mus81-Eme1 can cleave more efficiently Holliday junctions that are contained within larger regions of homology,
DNA, with a
312 bp homologous core, was used (Fig.
1A). The E. coli
Holliday junction resolvase RusA efficiently resolves this structure
generating four nicked duplex DNA species (Fig. 1B,
lane f). In comparison Mus81-Eme1 resolves
DNA
relatively inefficiently converting less than 5% of the available
substrate into duplex products at the highest concentration of enzyme
used (Fig. 1B, lane d).

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Fig. 1.
Cleavage of DNA by
Mus81-Eme1. A, schematic diagram of DNA
showing the Holliday junction situated within a central homologous core
(gray lines). The restriction sites used to generate the
structure from a figure-of-eight DNA molecule, the polarity of the DNA
strands, and the sizes (in kb) of each duplex arm are indicated.
B, agarose gel showing cleavage of the Holliday
junction in by Mus81-Eme1 and NLS-RusA-GFP to generate nicked
duplex species of the sizes indicated (in kb). The band or smear
running ahead of the 1.8-kb product band in lanes d and
f is probably generated by a pair of cleavages in strands of
unlike polarity so that one duplex arm is detached. The reactions in
lanes b-d contain 2, 10, and 20 nM Mus81-Eme1,
whereas lane f contains 20 nM
NLS-RusA-GFP.
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Mus81-Eme1 Cleaves a Replication Fork Substrate in the Leading
Strand Template--
To directly compare Mus81-Eme1's ability to
cleave Holliday junctions versus replication forks,
Kpn and
Sma substrates were generated by
restriction of
DNA in a common duplex arm at (
Sma)
or close (
Kpn) to the boundary with the homologous core
sequence (Fig. 2, A and
B).
Sma can either resemble a Holliday
junction or a replication fork depending on where its junction point
branch migrates. In contrast
Kpn retains a small
heterologous region at the end of the cleaved arm, which obstructs
formation of a replication fork substrate. Like
DNA,
Kpn is cleaved inefficiently by Mus81-Eme1 (Fig.
2C, lanes b-d). In contrast Mus81-Eme1 cleaves
Sma relatively well, generating 3.2- and 0.95-kb duplex
products (Fig. 2C, lanes g-i). The prevalence of
these cleavage products and relative absence of 2.4- and 1.75-kb duplex
bands indicates that Mus81-Eme1 has a strong bias toward cleaving
Sma in what would be the leading strand template if this
were a true replication fork (Fig. 2D). To see if this
cleavage bias is a conserved feature of homologues of Mus81-Eme1, we
purified S. cerevisiae Mus81-Mms4 and tested it on both
Kpn and
Sma (Fig. 2E).
Mus81-Mms4, like Mus81-Eme1, cleaves
Kpn
relatively poorly compared with
Sma (compare lanes
c and f) and generates almost exclusively 3.2- and
0.95-kb duplex products from its cleavage of
Sma
(lane f).

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Fig. 2.
Cleavage of a replication fork structure by
Mus81-Eme1 and Mus81-Mms4. A, schematic diagram of
DNA as in Fig. 1 but showing the position of restriction sites used
to generate Kpn and Sma, which are
positioned close to or at the border between the homologous core and
heterologous sequences, respectively. B, junctions
generated by KpnI and SmaI restriction of .
Both junctions are free to branch migrate within ~300 bp of
homologous sequence and are represented at the limit of their branch
migration toward cleaved KpnI or SmaI sites,
respectively. C, agarose gels of Kpn and
Sma cleavage by Mus81-Eme1. Sizes of cleavage products
(in kb) are indicated. The band running ahead of the 0.95-kb position
is the duplex arm generated by KpnI or SmaI
digestion of . Amounts of protein in lanes b-d are 2, 10, and 20 nM. D, schematic diagram of
Sma showing the approximate position of the Mus81-Eme1
cleavage site. E, agarose gels showing the comparison
between Mus81-Eme1 and Mus81-Mms4 for cleavage of Kpn
and Sma. Amounts of protein in lanes b,
c, e, and f are 30 nM.
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Mus81-Eme1 Cleaves Model Replication Forks 3-6 Bases 5' of the
Junction Point--
To more precisely determine the sites at which
Mus81-Eme1 and Mus81-Mms4 cleave fork substrates, a synthetic
replication fork called F1, made from short (~50 nucleotides)
oligonucleotides, was used (Fig.
3D). Four preparations of F1
were made, each labeled in a different oligonucleotide, and tested for
cleavage by Mus81-Eme1 and Mus81-Mms4 (Fig. 3A). Like
Sma F1 is cleaved by both Mus81-Eme1 and Mus81-Mms4 in
the equivalent of the leading strand template to detach one of its
duplex arms. Analysis of the reaction products on a denaturing gel
shows that both enzymes cut at the same sites (Fig. 3C,
lanes c and d), which map mainly to positions
between 3 and 6 bp 5' of the junction point in oligonucleotide 2 (Fig.
3D). More minor cut sites are also detected up to 11 bp 5'
of the junction point in the same oligonucleotide (Fig.
3C, lanes c and d and data not
shown). Slight differences in site preference between Mus81-Eme1 and
Mus81-Mms4 are apparent, as well as some minor cut sites that appear to
be exclusive to one or other of the enzymes (e.g. Fig.
3C, lanes n and o). To confirm that
the cleavage bias observed using F1 is not dictated by the nucleotide
sequence we constructed a further fork substrate called F2 (Fig.
3E). F2 consists of the same nucleotide sequences as F1 but
differs by the strand that is discontinuous at the junction point and
therefore which strand is assigned as the leading strand template. F2,
like F1, is cleaved by both Mus81-Eme1 and Mus81-Mms4 in the leading
strand template, the major cut sites being between 3 and 6 bp 5' of the
junction point in oligonucleotide 8 (Fig. 3B, C
and E). From these data we conclude that Mus81-Eme1 and
Mus81-Mms4 cleave replication fork substrates in a specific manner that
is directed by the structure of the substrate and seemingly independent
of its nucleotide sequence.

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Fig. 3.
Cleavage of F1 and F2 by Mus81-Eme1 and
Mus81-Mms4. A, native polyacrylamide gel showing
the cleavage of F1 by Mus81-Eme1 (Sp) and Mus81-Mms4
(Sc). The position of the 5' label (*) is shown in the
schematics of F1. B, as for A except the
substrate is F2. C, denaturing gel of some of the
reactions shown in A and B run beside G + A
sequencing ladders of the appropriate labeled oligonucleotide
(lane a, oligonucleotide 2; lane h,
oligonucleotide 8; lane l, oligonucleotide 5).
D, schematic of the central region of F1 with
arrows showing the position of the major Mus81-Eme1/Mms4
cleavage sites. Sp- and Sc-labeled arrows indicate cleavage
sites that are predominantly used either by S. pombe
Mus81-Eme1 or S. cerevisiae Mus81-Mms4 respectively.
E, same as D except that the junction is
F2.
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Cleaving Substrates That Resemble Different Types of Stalled and
Reversed Replication Fork--
It is hypothesized that a key role for
Mus81-Eme1 and Mus81-Mms4 is to cleave stalled and reversed replication
forks and thereby facilitate the break-induced restart of replication
(18, 19, 26). Blocked replication forks can in principle form a number of different structures depending on which template strand is blocked,
whether the lagging strand polymerase can uncouple from the leading
strand polymerase, and whether the fork reverses to allow the
re-pairing of template strands. The occurrence of these aberrant
structures in vivo has recently been confirmed by electron microscopy (30). To test Mus81-Eme1 and Mus81-Mms4 on structures that
resemble different types of stalled/reversed replication fork,
substrates F3 to F6 were made (Fig.
4A). F3 and F4 resemble replication forks that have reversed to expose either a 3' (leading nascent strand) or 5' (lagging nascent strand) single-stranded tail
respectively. F6 resembles a fork in which the lagging strand polymerase continued synthesis despite the leading strand being blocked. Finally, F5 resembles a normal replication fork since, unlike
the other substrates (including F1 and F2), it does not contain an
equivalent of the nascent lagging strand juxtaposed at the junction
point. Substrates F3, F4, and F6 are cleaved at least as well as F1 by
both Mus81-Eme1 and Mus81-Mms4 (Fig. 4A and data not shown).
In contrast little or no cleavage of F5 was detected (Fig.
4A, lanes n-p and data not shown). A comparison of the
rates of cleavage of these substrates shows that F3 and F6 are cleaved
at least 2-fold faster than either F1 or F4 (Fig. 4B).

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Fig. 4.
Comparing different types of stalled and
reversed forks for cleavage by Mus81-Eme1. A,
native polyacrylamide gel showing the cleavage of different fork
substrates by Mus81-Eme1. Substrates and reaction products are
indicated by the schematics, which include an asterisk to
show the position of the 5'-end label. Amounts of protein are 0.1 nM (lanes b, f, j,
n, and r), 1 nM (lanes
c, g, k, o, and
s), and 10 nM (lanes d, h,
l, p, and t). B,
rates of cleavage of fork substrates. Substrates are F1 (open
square), F3 (open circle), F4 (filled
circle), F5 (filled square), and F6 (filled
triangle).
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Mapping Cleavage Sites in F3, F4, and F6--
The same strategy
for mapping the cleavage sites in F1 and F2 was employed for
determining the cleavage sites in F3, F4, and F6. Like F1, both F3 and
F6 are cleaved predominantly in oligonucleotide 2 at positions 3-6 bp
5' of the junction point (Fig.
5A, lanes c and
g, B, and C and data not shown).
However, little or no cleavage in this strand is detected for F4 (Fig.
5A, lane e and data not shown). Instead F4 is
cleaved predominantly in oligonucleotide 5 4 bp 5' of the junction
point, which in this substrate is the equivalent of the lagging strand
template (Fig. 5A, lane l, D and data
not shown).

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Fig. 5.
Mapping the cleavage sites in F3, F4, and
F6. A, denaturing gel analysis of Mus81-Mms4
cleavage of F3, F4, and F6 labeled in either oligonucleotide 2 (lanes b-g) or 5 (lanes i-n). G + A sequencing
ladders of oligonucleotide 2 and 5 are in lanes a and
h, respectively. B-D, schematics of the
central regions of F3, F4, and F6. Arrows indicate the major
cleavage sites for Mus81-Eme1 and Mus81-Mms4.
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DISCUSSION |
Mus81-Eme1 and its homologue from S. cerevisiae
Mus81-Mms4 have both been implicated in cleaving DNA junctions at
stalled replication forks in order to facilitate some kind of
break-induced replication
(BIR)1 (18, 19, 26). Here we
have investigated the ability of recombinant SpMus81-Eme1 and
ScMus81-Mms4 to cleave various different DNA junctions that resemble
those that are expected to form at stalled replication forks following
complete or partial disassembly of the replisome. We confirm that these
recombinant enzymes cleave Holliday junctions relatively poorly
compared with three- or four-way DNA junctions that already contain
some kind of strand discontinuity at the junction point. Furthermore,
our side-by-side comparison of Mus81-Eme1 and Mus81-Mms4 shows that
these enzymes have the same substrate specificity. They also cut at the
same sites, albeit their preferred cleavage sites are in some cases
slightly different. These data have enabled us to speculate on the mode
of action of Mus81-Eme1/Mms4 in vivo (see below).
The Potential of Mus81-Eme1 and Mus81-Mms4 to Cleave Different
Types of Stalled and Reversed Replication Forks--
The stalling of
replication forks by depletion of dNTPs or blockage in the
DNA template appears to be a frequent event. What happens to the
replisome when it stalls is largely unknown. In many cases its
integrity and potential for further replication appears to be
maintained by cell cycle checkpoint kinases such as Rad53 in S. cerevisiae (31, 32). In the absence of RAD53 stalled
forks are converted into a range of abnormal structures, including
junctions with extensive single-stranded gaps, reversed forks with
extruded single-stranded tails, and reversed forks where the two
nascent strands have annealed to form a Holliday junction (30).
However, under certain circumstances, for example replication fork
blockage by a bulky lesion in the DNA, fork reversal may be actively
encouraged to provide room for DNA repair and/or a template for
replicative bypass of the lesion (10). Subsequently the DNA junction
formed by fork reversal would have to be removed and a means found to
restart replication. Our in vitro analysis of Mus81-Eme1 and
Mus81-Mms4 substrate specificity reveals how these enzymes are well
suited for processing most of the abnormal structures that can form
when the replisome disassembles (Fig. 6).
Reassuringly neither Mus81-Eme1 nor Mus81-Mms4 can efficiently cleave a
normal replication fork structure (Fig. 6A). A blockage in
the leading strand can result in the uncoupling of the two polymerases
(33) to generate the structure shown in Fig. 6B, i. This can be cleaved by Mus81-Eme1/Mms4 resulting in the
detachment of the leading strand arm (Fig. 6B,
ii). However, if fork reversal ensues before Mus81-Eme1/Mms4
has had a chance to act then the 5'-end of the nascent lagging strand
would be exposed to generate the structure shown in Fig. 6B,
iii. Mus81-Eme1/Mms4 would be able to cleave this structure
but incisions would be redirected to the lagging strand template
causing the detachment of the lagging strand arm. Mus81-Eme1/Mms4 would
also be able to cleave a reversed fork with the 3'-end of the nascent
leading strand exposed; however, here cleavage would occur in the
leading strand template resulting in the detachment of the leading
strand arm (Fig. 6C, ii-iii). However, if the
fork reverses to the point at which a Holliday junction is formed then
Mus81-Eme1/Mms4 may either act inefficiently or not at all (Fig.
6B, v and C, iv). This
suggests that there may be a limited window of opportunity for
Mus81-Eme1/Mms4 to cleave a replication fork undergoing reversal. Of
course even if a Holliday junction is formed its branch migration could
regenerate the substrates that are cleaved well by Mus81-Eme1/Mms4.

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Fig. 6.
Model of Mus81-Eme1/Mms4 cleavage of stalled
and reversed replication forks. Details of the model are discussed
in the main text. Nascent strands are represented as gray
lines with an arrowhead at each 3'-end. The star
indicates the position of the replication fork block. The position of
each Mus81-Eme1 cleavage site is shown and are essentially the same for
Mus81-Mms4. The dashed gray line in D indicates
that the 3'-tail is not drawn to scale with respect to the rest of the
DNA molecule.
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Why Cleave Stalled Replication Forks?--
Breaking a replication
fork, as envisaged in Fig. 6, B and C, is
potentially hazardous to the cell in at least two ways: (i) a single
non-repaired double-strand break can be lethal, and (ii) recombination,
which is necessary to repair the break, can be prone to generating
genomic rearrangements (34). This may be why enzymes like Rqh1 are so
important for genomic stability with their potential for resetting
reversed replication forks and thereby avoiding fork cleavage (11, 12,
14). Nevertheless, it is clear that in both prokaryotes and eukaryotes
recombination plays an important role in restarting replication at
non-origin sites (16, 17, 35, 36). In E. coli two models
have been invoked to explain how fork reversal, and the formation of a
Holliday junction, can be used to load recombinases at the stalled
fork. The first model says that the Holliday junction formed when the fork reverses is cleaved by RuvABC to generate a double-stranded end.
This end then acts as a substrate for RecBCD, which generates a 3'
single-stranded tail for RecA to load onto (6, 17, 35). Homologous
pairing and strand invasion then ensues to make a D-loop in which the
invading 3'-end primes leading strand synthesis, and where the PriA
protein can bind and orchestrate primosome reassembly for lagging
strand synthesis (37). The second model avoids the formation of a
single-end break by RecBCD processing the free DNA end formed by the
reversal of the fork. Subsequent invasion of the re-annealed parental
duplex can then occur before cleavage of the Holliday junction by
RuvABC (6, 17, 35). In eukaryotes, which of these pathways is used may
be inconsequential for genome stability since cohesion between sister
chromatids should ensure that collapsed forks are accurately repaired
by recombination (38). Nevertheless, if the second model is favored in vivo, then Mus81-Eme1/Mms4's bias for cleaving reversed
forks like F3 and F4 (i.e. in the parental strand that is
complementary to the exposed single-strand) means that junction
resolution will not collapse the fork (Fig. 6D). Clearly for
this to work cleavage would have to be delayed until strand invasion
had occurred. The interaction of S. pombe Mus81 with the
forkhead-associated-1 (FHA1) domain of Cds1 (a homologue of Rad53), and
its Cds1-dependent phosphorylation in the presence of
hydroxyurea (24), point to a potential way in which its cleavage
activity might be controlled.
Where Mus81-Eme1/Mms4 collapses the replication fork repair would
probably depend on BIR, although this may involve activities that are
distinct from normal BIR (26). In this respect it may be relevant that
the products of fork cleavage by Mus81-Eme1/Mms4 contain small
single-stranded gaps and 5' flaps (Fig. 6, B and C), and therefore would require further processing before
ligation. This means that a strand discontinuity could persist in the
intact chromatid that might play a role in directing the way in which the replication fork is repaired. Future studies will be required to
ascertain the validity of this idea as well as determining the types of
stalled replication forks that are actually targeted by Mus81-Eme1/Mms4
in vivo.