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J. Biol. Chem., Vol. 276, Issue 38, 35458-35464, September 21, 2001
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From the University of Texas Health Science Center and Institute of
Biotechnology, San Antonio, Texas 78245
Received for publication, June 13, 2001, and in revised form, July 6, 2001
Saccharomyces cerevisiae
RAD50 and MRE11 genes are required for the
nucleolytic processing of DNA double-strand breaks. We have
overexpressed Rad50 and Mre11 in yeast cells and purified them to near
homogeneity. Consistent with the genetic data, we show that the
purified Rad50 and Mre11 proteins form a stable complex. In the
Rad50·Mre11 complex, the protein components exist in equimolar
amounts. Mre11 has a 3' to 5' exonuclease activity that results in the
release of mononucleotides. The addition of Rad50 does not
significantly alter the exonucleolytic function of Mre11. Using
homopolymeric oligonucleotide-based substrates, we show that the
exonuclease activity of Mre11 and Rad50·Mre11 is enhanced for
substrates with duplex DNA ends. We have examined the endonucleolytic
function of Mre11 on defined, radiolabeled hairpin structures that also
contain 3' and 5' single-stranded DNA overhangs. Mre11 is capable of
cleaving hairpins and the 3' single-stranded DNA tail. These
endonuclease activities of Mre11 are enhanced markedly by Rad50 but
only in the presence of ATP. Based on these results, we speculate that
the Mre11 nuclease complex may mediate the nucleolytic digestion of the
5' strand at secondary structures formed upon DNA strand separation.
DSBs are induced by ionizing radiation and are also formed during
initiating events in various modes of homologous recombination (1).
Genetic studies in Saccharomyces cerevisiae have been instrumental in the discovery of genes required for recombination and
DSB1 repair through
homologous recombination. These genes, RAD50, MRE11, XRS2, RAD51, RAD52,
RAD54, RAD55, RAD57, RAD59,
and RDH54/TID1, are collectively referred to as the
RAD52 epistasis group (2).
A number of studies in S. cerevisiae indicate that DSBs in
homologous recombination events are processed in a nucleolytic fashion
to generate an intermediate with overhanging 3' ssDNA tails. The ssDNA
tails are bound by Rad51 and other recombination factors, which
function in concert to locate regions of homology on a corresponding
DNA duplex (a homologous chromosome or sister chromatid) and form
heteroduplex DNA joints (1, 2).
RAD50, MRE11, and XRS2 are involved in
the nucleolytic processing of DSBs. Analysis of the Rad50 and Mre11
sequences reveals homology of these two proteins to Escherichia
coli SbcC and SbcD, respectively (3), which combine to form a
complex that exhibits both exo- and endonuclease activities, including
the capacity to cleave hairpin structures (4). Mre11 from human and
yeast possesses exo- and endonuclease activities (5-9). Interestingly, overexpression of Rad50 and Mre11 can allow DNA synthesis to
efficiently progress through DNA sequences with a propensity to form
secondary structures, suggesting that the Mre11 complex in yeast might
also have the ability to cleave such DNA structures (10).
As evidenced by co-immunoprecipitation and two-hybrid experiments,
Rad50, Mre11, and Xrs2 are associated in a complex (7, 11). Likewise,
the human counterparts of these proteins, human Rad50, Mre11, and NBS1
(the Xrs2 equivalent) have also been purified from human cells as an
endogenous complex (8). Remarkably, genetic evidence in S. cerevisiae implicates RAD50, MRE11, and XRS2 in other cellular functions including the formation of
DSBs at meiotic recombination hotspots (1), non-homologous DNA
end-joining, and the maintenance of telomere length (1, 12). More
recently, Rad50 and Mre11 have been implicated in the adaptation to a
Rad9/Rad17-mediated G2/M checkpoint after the introduction
of an unrepairable DSB (13).
The multifunctional nature of the Rad50, Mre11, and Xrs2 trio
emphasizes the importance for the biochemical characterization of these
proteins, singly and in combination, for activities germane for their
biological functions. Here, we purify Rad50 and Mre11, reconstitute the
Rad50·Mre11 complex, and describe the endo- and exonucleolytic
activities of these factors. We speculate on how the DNA
structure-specific endonuclease activity of Rad50·Mre11 could be
utilized to create ssDNA tails that facilitate subsequent steps in
recombination, including the formation of dual Holliday junctions.
Overexpression of Rad50 and Mre11 in Yeast--
The
RAD50 and MRE11 genes were introduced into the
vector pPM231 containing the galactose-inducible hybrid
GAL-PGK promoter. The resulting plasmids, pR50.1 (2 µm,
GAL-PGK-RAD50, LEU-2d) and pM11.1 (2 µm, GAL-PGK-MRE11, LEU-2d), were
introduced into the protease-deficient strain BJ5464 (MAT Purification of Rad50--
All the steps were carried out at 0 to 4 °C. Typically, cells (50 g) expressing Rad50 were resuspended
in 75 ml of cell breakage buffer (50 mM Tris-HCl, pH 7.5, 10% sucrose, 10 mM EDTA, 1.2 M KCl, 1.0 mM dithiothreitol, and 0.01% Nonidet P-40) with protease inhibitors (5.0 µg/ml each of aprotinin, leupeptin, chymostatin, pepstatin A, and 1 mM each phenylmethylsulfonyl fluoride
and benzamidine) and lysed in a French press (SLM Aminco) at 20,000 p.s.i. The soluble fraction (Fraction I) was collected by
ultracentrifugation (100,000 × g, 90 min) and treated
with ammonium sulfate at 0.28 g/ml. The precipitate was collected by
centrifugation (17,000 × g, 25 min), redissolved in
100 ml of K buffer (20 mM KH2PO4, pH 7.4, 10% glycerol, 0.5 mM EDTA, 0.01% Nonidet P-40,
and 1.0 mM dithiothreitol) with protease inhibitors, and
dialyzed against K buffer + 100 mM KCl. The dialysate
(Fraction II) was applied to a 20-ml Q-Sepharose column, which was
developed over a 200-ml gradient from 100 to 540 mM KCl,
collecting 5.0-ml fractions. The peak fractions (about 350 mM KCl, Fraction III), as identified by Coomassie Blue
staining of SDS-PAGE gels, were pooled and applied directly onto an
8.0-ml Affi-Gel Blue (Bio-Rad) column, which was developed with an
80-ml gradient from 100 to 1800 mM KCl, collecting 2.0-ml
fractions. Peak elution occurred at about 1400 mM KCl, and
these fractions were pooled (Fraction IV), dialyzed against K buffer + 100 mM KCl, and fractionated in a 6.0-ml
macro-hydroxyapatite (Bio-Rad) column with a 50-ml gradient from 0 to
240 mM KH2PO4 in K buffer + 100 mM KCl, collecting 1.0-ml fractions. The peak fractions
(~175 mM KH2PO4, Fraction V) were
pooled, concentrated in a Centricon 30 concentrator to 0.7 ml, and
subjected to gel filtration on a 35-ml Sepharose 6B column in K buffer + 100 mM KCl. The peak of Rad50 (eluted at 16-22 ml,
Fraction VI) was further purified in a 1.0-ml Mono Q column using a
40-ml gradient from 100 to 370 mM KCl, collecting 1.0-ml
fractions. Rad50 (eluted at about 170 mM KCl, Fraction VII)
was concentrated in a Centricon 30 microconcentrator to a final volume
of 1.0 ml at 10 mg/ml. Rad50 was stored in small aliquots at
Purification of Mre11--
All the steps were carried out at
0-4 °C. Typically, extract (Fraction I) was prepared from yeast
cells (100 g) expressing Mre11 and subjected to ammonium sulfate
precipitation at 0.28 g/ml. The resulting precipitate was dissolved in
130 ml of K buffer with protease inhibitors and dialyzed against K
buffer + 100 mM KCl. The dialysate (Fraction II) was
applied to a 25-ml Q-Sepharose column developed with a 250-ml gradient
of 100-650 mM KCl, collecting 5.0-ml fractions. The peak
fractions of Mre11 (~300 mM KCl, Fraction III), as
identified by Coomassie Blue staining of SDS-PAGE gels, were combined,
bound to a 6.0-ml macro-hydroxyapatite column, and eluted with a
gradient of 0-270 mM KH2PO4 in K
buffer + 100 mM KCl. The Mre11 peak fractions from the
macro-hydroxyapatite step (~140 mM
KH2PO4) were pooled (Fraction IV), concentrated using a Centricon 30 concentrator to ~4.0 ml, and subjected to gel
filtration on a 200-ml Sephacryl 300 column in K buffer + 100 mM KCl. Mre11-containing fractions (eluted at 90-120 ml,
Fraction V) were loaded onto a 1.0-ml Mono S column, which was
developed with a 40-ml KCl gradient from 100 to 630 mM KCl,
with Mre11 eluting at about 300 mM KCl. The peak fractions
(Fraction VI) were concentrated in a Centricon 30 microconcentrator to
a final volume of 2.0 ml at 10 mg/ml. Purified Mre11 was stored at
Reconstitution of the Rad50·Mre11 Complex--
Fraction V
Rad50 (15 mg) was mixed with Fraction V Mre11 (15 mg) in 4.0 ml and
incubated on ice for 5 h to allow for complex formation. The
Rad50·Mre11 complex was separated from free Mre11 by gel filtration
on a 200-ml Sepharose 6B column equilibrated in K buffer + 100 mM KCl. Fractions were collected over a 200-ml volume, with
the Rad50·Mre11 complex eluting at 80-100 ml. The Rad50·Mre11
complex was further purified on a 1.0-ml Mono S column with a 40-ml
gradient (100-630 mM KCl), with the complex eluting at
~200 mM KCl. The peak fractions were pooled and
concentrated to a final volume of 1.0 ml at 10 mg/ml.
DNA Substrates--
Oligonucleotides were from Life
Technologies, Inc. TP8 (9), a 74-mer, had the sequence
(5'-GACCTGGCACGTAGGACAGCATGGGATCTGGCCTGTGTTACACAGTGCTACAGACTGGAACAAAAACCCTGCAG-3'). TP9 (9), also a 74-mer, was the exact complement of TP8. TP8E (73-mer)
was identical to TP8, except for the absence of the 3' G residue. T74
consisted of 74 T residues, and A74 consisted of 74 A residues. HP2
(56-mer) had the sequence
5'-AAAAAAGACCTGGCACGTAGGACAGCAGCTGCTGTCCTACGTGCCAGGTCAAAAAA-3', and
last, HP30 (84-mer) had the sequence
5'-AAAAAAGACCTGGCACGTAGGACAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCTGTCCTACGTGCCAGGTCAAAAAA-3'. These oligonucleotides were purified on 15% denaturing polyacrylamide gels containing 7.0 M urea and 20% formamide run at
55 °C in 100 mM Tris borate, pH 8.0, and 2.0 mM EDTA (TBE). The oligonucleotides were electroeluted from
gel slices, dialyzed against water, and concentrated in
Centricon 10 microconcentrators. The single-stranded oligonucleotide ladder used as size markers was purchased from Life
Technologies, Inc.
Radiolabeling of Nuclease Substrates--
DNA hairpin substrates
were prepared by labeling HP2 and HP30 at the 3' end with terminal
deoxynucleotidyltransferase and [
TP8, T74, and A74 were labeled at the 3' end with terminal
deoxynucleotidyltransferase and [ Nuclease Assays--
Various DNA substrates were mixed with
Mre11 or Rad50·Mre11 complex (amounts indicated in figure legends) in
reaction buffer (30 mM BisTris-HCl, pH 7.0, with 50 mM KCl, 2.5 mM MnCl2, 50 µg/ml bovine serum albumin, and 1.0 mM dithiothreitol). ATP or
ATP Thin Layer Chromatography--
Portions of the
exonuclease reactions were loaded onto polyethyleneimine plates
(J. T. Baker Inc.), which were developed in 1.0 M
formic acid and 0.3 M LiCl2. Reaction products
were visualized by autoradiography and quantitated by phosphorimage analysis.
Rad50, Mre11, and Rad50·Mre11 Complex--
SDS-PAGE analysis of
extracts from yeast cells harboring the Rad50 expression plasmid
(pR50.1) and the empty expression vector (pPM231) revealed the Rad50
protein band clearly. The observed size of the overexpressed Rad50
protein (150 kDa) (Fig. 1A,
left panel) was in agreement with the predicted molecular
mass for this protein (14). By immunoprecipitation, we have verified that Rad50 in wild type extract has the same gel size as the
overexpressed protein. Rad50 was purified (Fig. 1A,
middle panel) to near homogeneity (Fig. 1A,
right panel).
Likewise, comparison of extracts from yeast cells harboring the Mre11
expression plasmid, pM11.1, versus pPM231 showed the overexpression of Mre11 (Fig. 1B, left panel).
The apparent gel size of ~94 kDa for Mre11 was larger than the
predicted size of 78 kDa for this protein (11). By immunoprecipitation,
we have verified that the overexpressed Mre11 had the same gel size as Mre11 from wild type extract. Mre11 was also purified (Fig.
1B, middle panel) to near homogeneity (Fig.
1B, right panel).
When partially purified Rad50 and Mre11 were mixed on ice and applied
to a Sepharose 6B gel filtration column, the Mre11 profile was shifted
to co-elute with Rad50 as a higher molecular weight species (not
shown), suggesting that Rad50 and Mre11 formed a stable complex.
Interestingly, whereas Rad50 alone did not bind to a Mono S column, it
was efficiently retained after incubation with Mre11, which binds to
Mono S, thus providing additional evidence that these proteins form a
stable complex. For biochemical studies, the Rad50·Mre11 complex was
assembled and purified to near homogeneity as described under
"Experimental Procedures" and found to consist of equimolar amounts
of the two proteins (Fig. 1C, right panel). Neither the addition of ATP nor Mn2+ had any detectable
effect on complex stability (not shown).
Mre11 Exonuclease Activity Has 3' to 5' Polarity and
Has a Preference for Duplex DNA Ends--
To examine the exonuclease
activity of Mre11 and the Rad50·Mre11 complex, the 5'-labeled duplex
(5'-Duplex) and the 3'-labeled duplex (3'-Duplex) substrates were used.
Aside from the location of the 32P label, the 5'-Duplex and
the 3'-Duplex shared the same DNA sequence. To follow the kinetics more
accurately, the experiment was carried out at 4 °C. Digestion of the
5'-Duplex by either Mre11 or the Rad50·Mre11 complex as a function of
time generated a variety of radiolabeled products of decreasing size
(Fig. 2A). By contrast, digestion of the 3'-Duplex generated a single major product that migrated at the gel front (Fig. 2B). These results confirmed
that the exonuclease activity of Mre11 is 3' to 5' in polarity.
Likewise, the Rad50·Mre11 complex exhibited a 3' to 5' exonuclease
activity. In contrast to human Rad50, which stimulated the exonuclease
activity of human Mre11 3-4-fold (9), yeast Rad50 appears to have no significant effect on the exonuclease activity of Mre11. The
exonuclease product generated by the digestion of the 3'-Duplex by
Mre11 and Rad50·Mre11 was a mononucleotide, as determined by
comparison to the mobility of the mononucleotide product generated by
the dsDNA-specific 3' to 5' Exonuclease III from E. coli by
thin layer chromatography (Fig. 3) and on
polyacrylamide gels (not shown).
The exonuclease activity of Mre11 and Rad50·Mre11 required
Mn2+ (Fig. 3), which could not be substituted by
Mg2+, Ca2+, Zn2+, or
Co2+ (not shown). As expected, the exonuclease activity of
Mre11 was unaffected by ATP or ATP
We next examined whether Mre11 and Rad50·Mre11 also acted
exonucleolytically on ssDNA. For this purpose, oligonucleotide TP8 was
labeled at the 3' end with either an A or T residue, as described under
"Experimental Procedures." Incubation of these substrates with
Rad50·Mre11 (Fig. 5A) or
Mre11 (not shown) resulted in the release of the
32P-labeled nucleotides as observed for the 3'-Duplex
substrate. This result was in agreement with the observation of Usui
et al. (7). However, we also wished to consider the
possibility that hybridization of the ssDNA end in TP8 to short
complementary sequences within the oligonucleotide substrate or between
different TP8 molecules might transiently generate a duplex end for the
Rad50·Mre11 exonuclease function to act upon. To examine this, we
labeled the homopolymeric oligonucleotides T74 and A74, consisting of a
run of 74 T or A residues, at the 3' end with a labeled T or A residue,
respectively. Such substrates should be largely devoid of secondary
structure. As shown in Fig. 5B, Rad50·Mre11 had little ability to digest these homopolymers. Importantly, the hybrid generated
by the radiolabeled T74T* substrate with the unlabeled A74 substrate
(T*A Hybrid) was now digested by Rad50·Mre11 exonucleolytically (Fig.
5B), albeit less extensively than the 3'-Duplex (Fig.
2B). We attribute this difference to the fact that the TA
Hybrid is likely a population of annealed products, some of which have
persistent ssDNA overhangs or have the radiolabeled end embedded in
concatamers, thus preventing the labeled end from degradation. Similar
results were obtained using Mre11 alone (data not shown) and with
another unrelated enzyme, exonuclease III, which is a known
dsDNA-specific 3' to 5' exonuclease (Fig. 5C). Taken
together, the results demonstrated that the exonuclease activity of
Mre11 and Rad50·Mre11 has a clear preference for duplex DNA ends and
that digestion of the TP8 oligonucleotides as initially observed was
likely enhanced by transient base-pairing events that generated duplex
DNA ends.
DNA Structure-specific Endonuclease Activity of Mre11 and
Rad50·Mre11--
Both Mre11 (5-7) and Rad50·Mre11 (data not
shown) were capable of digesting circular plasmid-length ssDNA to
products of higher gel mobility, indicating the action of an
endonuclease. The fact that neither Mre11 nor Rad50·Mre11 cleaved a
homopolymeric oligonucleotide endonucleolytically (Fig. 5B)
suggested that they might be acting on secondary structures within the
ssDNA substrate. Therefore, it was of considerable interest to examine
the endonucleolytic function on defined hairpin substrates that mimic
structures expected in the ssDNA molecule. Difficulties in discerning
endo- versus exonuclease activities on such substrates were
overcome by the inclusion of a homopolymeric poly(dA) tail at the ends
of the hairpins, guided by our observation that such homopolymers were relatively resistant to exonucleolytic cleavage (Fig. 5B).
Interestingly, both Mre11 and Rad50·Mre11 were capable of making
specific incisions resulting in the creation of two major
endonucleolytic products, designated A and B. Product A was the result
of an incision at the distal end of the hairpin relative to the
3'-labeled extremity. Importantly, as determined by analysis of
reaction products on sequencing gels, the relative position of the
incision remained the same regardless of whether a fully paired hairpin
(HP2) or a hairpin with a 30-base homopolymeric loop (HP30) was used
(Fig. 6). Product B resulted from another
incision at the junction of the duplex with the 3' ssDNA poly(dA)
overhang (Fig. 6). Owing to the higher temperature (37 °C) and
increased incubation times (10-40 min) employed for these experiments
compared with the conditions (4 °C and 0.5-2.0 min) used to examine
the exonuclease activity of the homopolymeric substrates in Fig.
5B, a modest amount of mononucleotide (AMP*) was
observed (Fig. 6).
Effect of ATP on the Endonuclease Function of
Rad50· Mre11--
We addressed the question of whether ATP was
important for the structure-specific endonucleolytic function of Mre11
and Rad50·Mre11. In the absence of ATP, Rad50·Mre11 was very
similar to Mre11 with regard to overall endonuclease activity (Fig.
7, lanes 5 and 11). However, in the presence of ATP, the endonuclease activity of the
Rad50·Mre11 complex was greatly enhanced, whereas that of Mre11 was
unaffected (Fig. 7, lanes 3 and 9). For instance,
whereas only 11% of HP2 was converted to products A and B by
Rad50·Mre11 after 40 min in the absence of ATP (Fig. 7, lane
11), ~52% was converted into these products when ATP was
present (Fig. 7, lane 9). Importantly, ATP Exonuclease Activity of Mre11 and
Rad50·Mre11--
Mre11 and Rad50·Mre11 exhibit dsDNA exonuclease
activity with a 3' to 5' polarity that releases mononucleotide
products. Although neither Mre11 nor Rad50·Mre11 are significantly
stimulated by ATP for its exonuclease function, the activity of
Rad50·Mre11 is inhibited by ATP DNA Structure-specific Endonucleolytic Activity of
Mre11 and Rad50·Mre11--
Based on our finding that the exonuclease
activity of Mre11 was attenuated by homopolymeric ssDNA sequences, we
designed hairpin structures with poly(dA) extensions to allow us to
discern the endonuclease function. Using this approach, we were able to
observe two distinct endonucleolytic activities within hairpin
substrates. One of the endonucleolytic products was formed upon
cleavage at the distal end of a hairpin loop relative to the labeled 3'
end. The second major endonucleolytic product was formed upon cleavage at the junction between the duplex DNA molecule and the 3' ssDNA extension. Although Mre11 was capable of making both incisions alone,
Rad50 stimulated these activities markedly, but ATP was specifically
required for the maximal expression of the endonucleolytic activities
of only the Rad50·Mre11 complex. Similar DNA structure-specific endonuclease activities were first reported by Paull and Gellert (15)
for the human Mre11 protein, but our current results point to two major
differences concerning how these endonucleolytic activities in the
yeast and human counterparts are regulated. Specifically, whereas the
ability to incise a fully paired DNA hairpin and a 3' ssDNA tail for
hMre11 is largely dependent on both the human Rad50 and NBS1 proteins
(15), S. cerevisiae Rad50 alone is sufficient to
stimulate these activities in yeast Mre11, although it remains possible
that Xrs2 may further enhance the endonucleolytic function of
Rad50·Mre11. Second, although the incision of a 3' ssDNA tail but not
a DNA hairpin by the human Rad50·Mre11·NBS1 complex requires ATP,
the yeast Rad50·Mre11 complex needs ATP for the maximal expression of
both structure-specific activities.
The Mre11 Endonucleolytic Function and DSB Processing--
Results
from a number of genetic studies in S. cerevisiae indicate
that during DSB processing, the DNA strands that contain the 5' termini
are resected preferentially (1). Given this observation, it is clear
that the Mre11 exonuclease activity, which has a 3' to 5'
directionality, cannot be solely responsible for the resection of the
5' DNA strands. Like its human counterpart, yeast Mre11 also possesses
the ability to incise DNA hairpin structures as well as 3' ssDNA tails
that border a duplex region. The highly conserved nature of these
endonucleolytic activities suggests that they are germane for the
processing of DNA DSBs. We speculate that these DNA structure-specific
activities may be used in conjunction with a DNA helicase to process
DNA double-strand breaks. The engagement of the DNA ends by the
Rad50·Mre11·Xrs2 complex and recruitment of a DNA helicase would
lead to catalytic unwinding of the two DNA strands, creating 3' and 5'
single-stranded overhangs (Fig. 8A, Step I). We
also surmise that this helicase may be activated by a ssDNA region
generated by the 3' to 5' exonuclease activity of the complex or
through the limited capacity of Rad50 to partially unwind duplex DNA
(15). As postulated in Fig. 8A, the endonucleolytic function
of the Rad50·Mre11·Xrs2 complex may recognize and incise secondary
structures present in the DNA strands that harbor the 5' termini of the
DNA break (Fig. 8A, Step II). This hypothetical model (Fig. 8) resembles the mode of DSB processing in E. coli, during which the ends of DSBs are acted upon by the
integrated helicase/nuclease action of the RecBCD protein
complex (16).
The end result of such a mode of processing would in fact create not
only a 3' ssDNA tail but also a shorter 5' ssDNA tail as well (Fig.
8A, Step III). Given that Rad51 can efficiently utilize both 3' and 5' ssDNA tails for DNA joint formation (17), the
DNA intermediate depicted in Fig. 8A (III) is
ideally suited for the formation of double Holliday junctions (Fig.
8B) known to exist during meiotic recombination (1).
At this time, we can only speculate as to the reason(s) why secondary
structures within the 3' strand are not also acted upon by the
Rad50·Mre11·Xrs2 complex. Genetic and biochemical evidence clearly
indicates that either the 3' strand is protected or that the 5' strand
is preferentially excised. We suggest that the 3' ssDNA generated as
result of the helicase action might be subject to preferential binding
by a variety of proteins that would, in turn, function to protect the
3' ssDNA tail and/or melt the secondary structures that might otherwise
be susceptible to the action of Rad50·Mre11·Xrs2. Among such
protein factors might be RPA, Rad51, and other recombination factors
that have been shown to localize to the site of a DSB (18-20). We wish
to emphasize that the proposed model is suggestive and must be
validated experimentally.
Functional Redundancy of the Mre11 Nuclease in Mitotic
cells--
Point mutations that render Mre11 nuclease deficient cause
no apparent anomalies in DSB processing in mitotic cells. We would imagine that the most important role of the Rad50·Mre11·Xrs2
complex in end-processing in such instances is to recruit a DNA
helicase to the DSB. Once a branched DNA structure is generated as a
result of catalytic DNA strand separation by the helicase (Fig.
8A, Step II), the fact that nuclease-deficient
mutants of Mre11 are still capable of DSB processing (21) would argue
that the 5' ssDNA strand in the branched structure is also eventually
accessible to other nucleases, as suggested by the work of
Symington et al. (21).
It has been shown that in the absence of Mre11, DNA ends can still be
processed, perhaps by a dsDNA exonuclease (21). However, the low
processivity of 5' strand resection in such instances is evidenced by
highly reduced conversion tract lengths (21). This observation strongly
implicates the Rad50· Mre11·Xrs2-dependent pathway as the
preferred mechanism for DSB processing in wild type cells.
We thank Sabrina Stratton for technical
assistance and Deanna Jansen for helpful discussions.
*
This work was supported by National Institutes of Health
Research Grants R01 ES07061 and R01 GM57814.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, July 13, 2001, DOI 10.1074/jbc.M105482200
The abbreviations used are:
DSB, double-strand
break;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
ATP
DNA Structure-specific Nuclease Activities in the
Saccharomyces cerevisiae Rad50·Mre11 Complex*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ura3-52 trp1 leu2
1 his3200 pep4::HIS3 prb11.6R can1).
Cultures were grown overnight to the stationary phase in synthetic
medium lacking leucine and diluted 8-fold into leucine dropout
synthetic media with 3% glycerol, 3% lactic acid, and 2% galactose.
After 24 h of growth at 30 °C, cells were harvested by centrifugation.
80 °C. The Rad50 protein concentration was determined by
densitometric scanning of a 7.5% SDS-PAGE gel containing multiple
loadings of purified Rad50 against known amounts of bovine serum
albumin run on the same gel.
80 °C in small aliquots. The Mre11 protein concentration was
determined by densitometric scanning of a 7.5% SDS-PAGE gel containing
multiple loadings of purified Mre11 against known amounts of bovine
serum albumin run on the same gel.
-32P]ddATP. The
labeled oligos were held at 70 °C for 10 min and then allowed to
cool slowly to room temperature to anneal. The annealed product was
further purified on a 10% native polyacrylamide gel in TBE at 4 °C
and recovered by electroelution. The 5' end-labeled duplex substrates
were made by hybridizing oligo TP8 (10 µg) radiolabeled at the 5' end
with T4 polynucleotide kinase and [
-32P]ATP to oligo
TP9 (15 µg) held at 70 °C in 30 mM Tris HCl, pH 7.0, with 200 mM KCl for 10 min, which was allowed to slowly
cool to room temperature. Substrate concentrations were determined by
titration on a 10% native polyacrylamide gel in TBE against known
amounts of DNA standards and by scintillation counting. The 3'
end-labeled duplex substrates were generated by the hybridization of
oligo TP8E to TP9 and subsequent filling-in of the resulting single-base overhang with the exonuclease-deficient Klenow polymerase and [-32P]dGTP. The labeling reaction was halted by
phenol/chloroform extraction followed by gel filtration in Biospin P-6
columns (Bio-Rad) to remove the unincorporated isotope.
-32P]dATP or
[-32P]dTTP where indicated. TP8A* indicates that the
TP8 substrate (74-mer) was labeled at the 3' end with an A, and TP8T*
indicates 3'-labeling with a T. For these reactions, a molar excess of
oligonucleotide to isotope was used to minimize the possibility that
multiple bases were added to a single oligonucleotide. The T*A hybrid
was obtained by hybridizing T74T* to A74.
S (1.0 mM) were also added when stated. After
incubation at 37 or 4 °C where indicated, the reaction was halted by
1/10 volume of 3% SDS and deproteinized with proteinase K (0.5 mg/ml)
at 37 °C for 10 min. For oligonucleotide-based nuclease assays,
one-third volume of 90% formamide with 0.05% bromphenol blue was
added to each sample, which was boiled for 2 min before loading 15%
denaturing polyacrylamide gels containing 7 M urea and 20%
formamide in 100 mM Tris borate, pH 8.0, and 2.0 mM EDTA (TBE). After electrophoresis, the gels were fixed
in 10% acetic acid, 10% methanol, dried, and subjected to
autoradiography and phosphorimage analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Rad50 and Mre11 are overexpressed, purified,
and reconstituted as a complex. A, SDS-PAGE analysis of
extracts from yeast harboring the empty expression vector pPM231
versus cells transformed with pR50.1 (left panel,
lanes 1 and 2). The middle panel
depicts the purification scheme for Rad50. Purified Rad50 (1.0 µg)
was run on a 7.5% SDS-PAGE gel and stained with Coomassie Blue
(right panel). B, SDS-PAGE analysis of extracts
from yeast harboring pPM231 or pM11.1 (left panel,
lanes 1 and 2). The middle panel
depicts the purification scheme for Mre11. Purified Mre11 (1.0 µg)
was run on a 7.5% SDS-PAGE gel and stained with Coomassie
Blue (right panel). C, purification scheme
for the Rad50·Mre11 complex (left panel). Purified
Rad50·Mre11 complex (2.0 µg) was run on a 7.5% SDS-PAGE gel and
stained with Coomassie Blue (right panel). M,
mass standard.
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Fig. 2.
Mre11 and the Rad50·Mre11
complex have 3' to 5' exonuclease activity. A, the
5'-labeled duplex substrate (5' Duplex, 2.0 pmol) was
incubated at 4 °C with 15.0 pmol of Mre11 or Rad50·Mre11
(R/M) in 50 µl. At the indicated times, 8.0-µl aliquots
were removed, deproteinized, and run on a 15% denaturing
polyacrylamide gel at 55 °C. B, nuclease reactions were
performed on the 3'Duplex using the same conditions in
A.
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Fig. 3.
The exonuclease activity of Mre11
and the Rad50·Mre11 (R/M) complex is dependent on
Mn2+. The exonuclease activity
of Mre11 and Rad50·Mre11 on the 3' Duplex was monitored in the
presence of Mg2+ or Mn2+, as shown. Reactions
were carried out as described in Fig. 2 for 2 min and were analyzed by
thin layer chromatography. GMP and the reaction products generated by
exonuclease III were run as standards. GMP*, guanosine
monophosphate.
S. Interestingly, whereas ATP was
clearly not a requirement for the Rad50·Mre11 exonuclease function,
ATPS caused partial inhibition (Fig.
4, A and B). For
example, whereas ~73% of the labeled nucleotide was released from
the duplex by Rad50·Mre11 with or without ATP in 2 min, only ~36%
was liberated in the presence of ATPS (Fig. 4, A and
B). These results indicated that the ATPS-bound form of
Rad50·Mre11 is less active exonucleolytically. Other results
indicated that Rad50 and the Rad50·Mre11 complex possessed only a low
level of ATPase activity with kcat values of
0.10 min
1 and 0.12 min1 at 37 °C,
respectively. The exonuclease activity of Mre11 and Rad50·Mre11 was
maximal from pH 6.4 to 7.2 and was considerably less active above pH
7.6. As expected, Rad50 alone was devoid of any exonuclease
activity (not shown).
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Fig. 4.
The exonuclease activity of
Rad50·Mre11 is attenuated by ATP S.
A, the exonuclease activity of the Rad50·Mre11 complex was
monitored for its dependence on ATP. Reactions containing the 3' Duplex
were carried out as described for Fig. 2 for the indicated times and
were analyzed by thin layer chromatography. GMP*, guanosine
monophosphate. B, graphical representation of data from
A. Open squares, +ATP; filled squares,
ATP; closed circles, +ATPS.
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Fig. 5.
The exonuclease activity of Rad50·Mre11 has
a preference for duplex DNA ends. Various oligonucleotide
substrates (0.8 pmol each) were incubated with Rad50·Mre11 (6.0 pmol)
in 20 µl at 4 °C. A: closed squares,
TP8A*; open squares, TP8T*. B:
open triangles, T*A hybrid; open circles, T74T*;
filled circles, A74A*. C, same as B
but with exonuclease III.
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Fig. 6.
Mre11 and Rad50·Mre11 have DNA
structure-specific endonuclease activities. The fully paired
hairpin, HP2, and the hairpin with a 30-base loop, HP30, 4.5 pmol each,
were incubated at 37 °C with Mre11 or Rad50·Mre11 (11.0 pmol) in a
volume of 25 µl with 1.0 mM ATP. At the indicated times,
5.0-µl aliquots were deproteinized and run on denaturing
polyacrylamide gels. A and B mark the sites of
specific incisions. AMP* denotes the mononucleotide product
of remnant exonuclease activity.
S did not
stimulate the endonuclease activity of Rad50·Mre11 (Fig. 7,
lanes 6 and 12), nor did ATPS have any influence on the endonucleolytic function of Mre11. As expected, the
structure-specific endonuclease activities had a specific requirement
for Mn2+ as cofactor (Fig. 7).
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Fig. 7.
ATP stimulates the structure-specific
endonuclease activity of Rad50·Mre11. HP2 was incubated with
either Mre11 or Rad50·Mre11 as described in Fig. 7. Where indicated,
MnCl2 was omitted or substituted with MgCl2,
and ATP (1.0 mM) was omitted or replaced with ATP S.
Portions (5.0 µl) of each sample were removed after 20-min
incubations at 37 °C, deproteinized, and analyzed.
A and B mark the sites of specific
incisions. Bl, blank (no protein); Me, no metal
cofactor; S, ATPS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. Interestingly, whereas hRad50
has been shown to stimulate the exonuclease activity of human Mre11
(9), we find that S. cerevisiae Rad50 does not appear
to significantly affect the exonuclease activity of S. cerevisiae Mre11. We also demonstrate that Mre11 with or without
Rad50 has a clear preference for duplex DNA ends. The apparent ability
of Mre11 to digest ssDNA substrates as seen by Usui et al.
(7) was likely enhanced by the formation of transient duplex ends
within the oligonucleotide substrates.
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Fig. 8.
Models. A, hypothetical model
for DSB processing. The Rad50·Mre11·Xrs2 complex (R/M/X)
is postulated to recruit a DNA helicase for the catalytic unwinding of
the DNA duplex (I) to create a branched DNA structure
(II). It is suggested that the endonucleolytic activity of
the Rad50·Mre11·Xrs2 complex acts preferentially on secondary
structures in the 5' ssDNA strand in the branched intermediate to
create a 3' ssDNA tail and a shorter 5' ssDNA tail (III).
Gray slashes denote protection of the 3' ssDNA strand from
degradation. B, formation of dual Holliday junctions. Based
on the known properties of Rad51 and its accessory factors (marked by
the shaded ovals) (17), we suggest that the product of DSB
processing is utilized for the formation of double Holliday junctions
as depicted.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: University of Texas
Health Science Center and Institute of Biotechnology, 15355 Lambda Dr.,
San Antonio, TX 78245. E-mail: sung@uthscsa.edu.
ABBREVIATIONS
S, adenosine 5'-O-(thiotriphosphate);
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA;
PAGE, polyacrylamide
gel electrophoresis;
TBE, Tris borate EDTA.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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