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Previous Article | Next Article 
Molecular and Cellular Biology, November 1999, p. 7681-7687, Vol. 19, No. 11
Laboratory of Genetics, University of
Wisconsin Medical School, Madison, Wisconsin 53706
Received 16 June 1999/Returned for modification 30 July
1999/Accepted 19 August 1999
Saccharomyces cerevisiae mre11 Repair of DNA double-strand breaks
(DSBs) by homologous recombination requires a sister chromatid or a
homologous chromosome as a template. In Saccharomyces
cerevisiae, the sister chromatid is the preferred template for the
repair of damaged DNA (20). Consequently, ionizing radiation
(IR) resistance of wild-type haploid and diploid cells is maximal
in the G2 phase of the cell cycle when sister
chromatids are present (8, 9). However, wild-type
diploid strains are more resistant to IR-induced DNA damage than
haploid strains, reflecting that chromosomal homologues can also serve
as templates for repair (28, 33).
Genetic and biochemical analyses have implicated the Mre11-Rad50-Xrs2
protein complex in nonhomologous end joining (NHEJ) (14, 21, 29,
31). The rate of spontaneous heteroallelic recombination is
increased in mre11 We tested this hypothesis by measuring cell survival of synchronous
cultures following irradiation and by scoring sister chromatid recombination (SCR) and interhomologue recombination genetically. We
found that Mre11 deficiency leads to a decrease in homologous recombination-based DSB repair. Both SCR and interhomologue homologous recombination are affected by Mre11 deficiency, but the defect is most
pronounced with respect to SCR. Since SCR and interhomologue recombination were normal in hdf1 The phenotypic features of mre11 Yeast strains.
The genotypes of yeast strains used in this
study are listed in Table 1.
MATa/
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous
Recombination-Based Double-Strand Break Repair in
Saccharomyces cerevisiae
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mutants are
profoundly deficient in double-strand break (DSB) repair, indicating
that the Mre11-Rad50-Xrs2 protein complex plays a central role in
the cellular response to DNA DSBs. In this study, we examined the role
of the complex in homologous recombination, the primary mode of DSB
repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue
recombination genetically. mre11 strains were profoundly
sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant
strains exhibited decreased frequencies of IR-induced sister chromatid
and interhomologue recombination, indicating a general deficiency in
homologous recombination-based DSB repair. Since a nuclease-deficient
mre11 mutant was not impaired in these assays, it appears
that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein
complex in facilitating homologous recombination is independent of its
nuclease activities.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, rad50, and
xrs2 diploid strains relative to that of the wild type
(1), indicating that deficiency in the complex does not
abrogate homologous recombination. However, the extent to which DSB
repair is impaired in mre11 mutants (7, 26)
suggests that the impact of Mre11 deficiency extends beyond NHEJ.
Therefore, we hypothesized that mre11 mutants are
deficient in homologous recombination and that this defect results from
a diminished ability to utilize the sister chromatid as a template for
recombinational DNA repair.
mutants, the defects
observed in mre11 strains are not a general feature of
NHEJ mutants. Further, the data indicate that the nuclease activity of
the S. cerevisiae Mre11-Rad50-Xrs2 protein complex is not
required for homologous recombination, as SCR and interhomologue
recombination were normal in a nuclease-deficient mre11 strain.
mutants described herein
are consistent with the hypothesis that the S. cerevisiae
Mre11-Rad50-Xrs2 protein complex stabilizes chromatid interactions, and
thus plays a structural role in the homologous recombination process.
The data also suggest that the complex may regulate resection of DSB ends to facilitate homologous recombination.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
diploid strains JPY145, JPY146, JPY260, and
JPY264, capable of arresting the cell cycle in response to the mating
pheromone 
-factor, were constructed by transformation of strains
JPY41, JPY45, JPY84, and JPY259, respectively, with plasmid pFP18 (a
gift of Jim Haber) linearized with PvuII. Integrative transformation of this construct results in replacement of the HO-site-containing 138-bp BglII/BsaAI fragment of
the MAT locus with the hisG-URA3-hisG cassette
(2). Disruption was assessed morphologically by response to
-factor and was confirmed by Southern blotting. The
hdf1 disruption was introduced into diploid strains JPY45, JPY67, and JPY115 using plasmid pHSX-YKuLEU2 as described (5). Double mutant haploids were obtained by sporulation and tetrad dissection of double heterozygotes. For XRS2
disruption in diploid strain JPY115 by one-step gene replacement, a
hisG-URA3-hisG cassette (2) was inserted into the
HincII/BglII site in XRS2, deleting
all but 213 bp at the 5' end and 383 bp at the 3' end of the coding
sequence.
TABLE 1.
Yeast strains used in this study
200 mutation in JPY102 deletes HIS3
sequences contained in the SCR construct so that histidine prototrophy
can be generated only by an unequal SCR event. All strains were
cultured at 30°C. Disruptions were confirmed by Southern blotting.
Yeast media were prepared and strain manipulations were carried out
according to standard procedures (4, 13).
Plasmids. For construction of pScM11-314, a 2.9-kb BamHI/KpnI MRE11 fragment was subcloned from pSK-MRE11-BNX (pSK-ScMRE11 [7] digested with NruI and XhoI, blunted, and reclosed, deleting sequences 3' of the MRE11 stop codon) into the centromeric vector pRS314 (34). MRE11 expression from this construct is under the control of the native MRE11 promoter. The ADH1 promoter-driven MRE11 and mre11-3 expression constructs have previously been described (7).
Cell cycle arrest.
Cultures were grown to a density of
approximately 5 × 106 cells/ml and arrested in
G1 with -factor (3 µM final concentration, incubated
for 2 to 3 h) (U.S. Biologicals) or in G2 with
carbendazim (150 µg/ml final concentration, incubated for 2 to 3 h) (Aldrich). Synchronization was assessed morphologically and by flow
cytometry (see below).
Flow cytometry. Cells were fixed in 70% ethyl alcohol at 4°C for at least 12 h, were pelleted, were resuspended in 1 ml of 50 mM sodium citrate (pH 7.5), and were sonicated for 15 s. Cells were resuspended in sodium citrate containing 0.25 mg of RNase A per ml, were incubated at 50°C for 1 h or at 37°C overnight, and were resuspended in sodium citrate containing 1 µM Sytox Green (1:5,000 dilution) (Molecular Probes). Samples were kept in the dark at room temperature for at least 1 h prior to flow cytometric analysis.
Irradiation studies. Strains were irradiated in mid-log phase (approximately 107 cells/ml) or following cell cycle arrest (see above) as previously described (7).
Sister chromatid recombination. To monitor spontaneous SCR, approximately 100 cells from an overnight culture were used to inoculate fresh 50-ml yeast extract-peptone-dextrose (YEPD) cultures, and the cultures were then grown to a density of approximately 5 × 107 cells/ml. Approximately 5 × 107 cells per synthetic complete (SC) medium plate lacking His (SC-His) and 500 cells per nonselective plate were plated in triplicate. The rate of spontaneous SCR was determined from at least nine independent cultures per strain by fluctuation analysis (23) with modifications as previously described (7).
To measure IR-induced SCR, cultures were grown to mid-log phase (approximately 107 cells/ml) in YEPD. Approximately 3 × 108 cells were harvested for each strain and were resuspended in 900 µl of double-distilled water (ddH2O). Each cell suspension was split into two aliquots, one of which was irradiated on ice with 50 Gy while the other served as the unirradiated control. Cells were then diluted 10-fold into fresh YEPD, were allowed to recover for 30 min at 30°C, and were plated as described above. The number of IR-induced SCR events was determined by subtracting the ratio of histidine prototrophs to total viable cells in the unirradiated sample from the same value in the irradiated sample.Interhomologue recombination. To measure IR-induced interhomologue recombination, cultures of JPY264 transformants were grown to early log phase (approximately 5 × 106 cells/ml) in SC-Trp-Met media. Cultures were then split into three aliquots for asynchronous and G1- and G2-synchronized samples as described above. Each was then resuspended in ddH2O and split into three aliquots, two of which were irradiated on ice with 50 Gy and 150 Gy while the third served as the unirradiated control. Cells were then diluted 10-fold into fresh SC-Trp media, were allowed to recover for 30 min at 30°C, and were plated onto SC-Trp and SC-Trp-Met canavanine plates. The number of IR-induced interhomologue recombination events was determined by subtracting the ratio of canavanine-resistant methionine prototrophs to total viable cells in the unirradiated sample from the same value in the irradiated sample.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Synchronous cultures of haploid and diploid strains provide a
means to examine the ability of cells to utilize different homologous templates for recombinational DSB repair. In G1-synchronous
cultures, cells must rely on NHEJ to repair DSBs in the absence of
homology (Fig. 1). Haploid cells contain
a homologous template, in the form of a sister chromatid, only during
the G2 phase. In contrast, diploid cells contain homologous
templates, in the form of homologous chromosomes, throughout the cell
cycle. Additionally, sister chromatids are present in the
G2 phase and are the preferred template for repair
(20). We monitored the contribution of each of these templates to the survival of mre11 cells following
irradiation using synchronous cultures of haploid and diploid strains.
|
Radiation sensitivity of haploid mre11 strains.
Given the DSB repair deficiency observed in mre11 strains
(7, 26), we asked whether homologous recombination was
impaired by Mre11 deficiency, as suggested by previous work using
rad50 and xrs2 strains (18). In
order to determine whether mre11 strains were defective
in SCR, we examined the IR sensitivity of synchronous cultures of
wild-type (JPY70) and mre11 (JPY69) haploid strains.
Cells were grown to early log phase and arrested in G1 or
G2 by treatment with -factor or carbendazim,
respectively. Synchronization of cultures was confirmed morphologically
and by flow cytometry. Cells were irradiated in suspension at 150 Gy
and were plated onto rich media to score cell survival relative to that
of unirradiated cultures.
haploid strain JPY69 exhibited 0.7% survival (Fig.
2). Whereas wild-type cells arrested in
G1 exhibited 1.5% survival, cells in G2
exhibited 70% survival, indicating that the presence of a sister
chromatid in G2-synchronous cultures increases the survival of wild-type cells.
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cells did
not increase survival following IR. G2-synchronous
mre11 cells exhibited less than a twofold increase in
survival upon irradiation relative to that of
G1-synchronous cells (1 and 0.6% survival, respectively) (Fig. 2). Hence, the IR sensitivity of asynchronous haploid
mre11 strains can primarily be attributed to the
increased sensitivity of the G2 population. These data
suggest that use of the sister chromatid for recombinational repair is
substantially reduced in mre11 mutants.
The IR sensitivity of wild-type haploid cells in G1
demonstrates that NHEJ does not contribute significantly to cell
survival following IR. Indeed, a substantial fraction of the surviving cells are unlikely to have received any DSBs at the IR dose used. A
dose of 150 Gy is predicted to impart four to six DSBs per cell (32). Since IR-induced breaks are distributed
stochastically, 0.25 to 2% of cells were predicted to sustain less
than one DSB at the level of irradiation employed in this experiment
a
dose at which we observed approximately 1.5% survival. Hence, most of
the surviving cells in the G1-synchronous populations may
not have sustained DSBs.
Radiation-induced sister chromatid recombination.
To assess
SCR genetically, we used a chromosomal substrate consisting of a
tandemly repeated HIS3 gene in which the first repeat is
truncated at the 5' end and the second is truncated at the 3' end
(11). In this configuration, a functional HIS3 gene can be generated only by an unequal SCR event (Fig.
3). The wild-type (JPY92 and
JPY202) and mre11 (JPY97 and JPY98) haploid strains
exhibited similar rates of spontaneous SCR ([1.4 to 2.0] × 10
6 per generation).
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6 (Table
2). We consistently observed
significantly smaller increases in SCR frequency following irradiation
of the mre11 strains JPY97 and JPY98 (4.8 × 106). Similar data were obtained with a mutant of another
member of the complex. The xrs2 strains JPY155 and JPY156
exhibited an IR-induced increase in SCR frequency of 5.4 × 106 (Table 2). At this IR dose, the decrease in survival
of these mutant strains relative to the survival of the wild type
(1.5-fold) is of similar magnitude to the decrease in the number of
recombinants scored (2.6-fold).
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strains are
deficient in NHEJ (14, 21, 31). We asked whether the
decrease in SCR in mre11 cells was a general, and
presumably indirect, outcome of NHEJ deficiency. We analyzed the IR
sensitivity of synchronous cultures and the IR-induced frequency of SCR
in the haploid hdf1 strains JPY181, JPY174, and JPY176,
which are also impaired in NHEJ (6, 25). Haploid
hdf1 cells were no more IR sensitive than wild-type
cells, exhibiting survival of 38% in asynchronous cultures, 1.5% in
G1-synchronous cultures, and 81% in
G2-synchronous cultures (Fig. 2). The rate of spontaneous
SCR in the haploid hdf1 strains JPY174 and JPY176 was
1.5 × 106 per generation, not significantly
different from those of the other mutant and wild-type strains examined
in this assay (data not shown). However, in contrast to the
mre11 and xrs2 strains, the
hdf1 strains showed an IR-dependent increase in SCR
frequency equivalent to that of the wild type (16.3 × 106) (Table 2).
The IR sensitivity of haploid mre11 cells was partially
suppressed by the hdf1 mutation. Survival of the
mre11 hdf1 strain JPY254 was 5.7-fold greater than
that of the mre11 strain JPY69 following irradiation of
asynchronous cultures, with G1- and
G2-synchronous cultures displaying 2.2- and 4.6-fold
increases in survival, respectively (Fig. 2).
Radiation sensitivity of diploid mre11 strains.
We previously showed that diploid mre11 strains exhibit
higher radiation resistance than their haploid counterparts
(7), consistent with the behavior of rad50 and
xrs2 strains (18). As in wild-type cells, this
effect is presumably due to the presence of homologous chromosomes for
repair of DSBs (33). To assess the ability of
Mre11-deficient diploid cells to use interhomologue recombination for
DSB repair,
MATa/mat::hisG
disruptions were established in wild-type (JPY41) and
mre11 (JPY45) diploid strains to allow G1
synchronization with -factor. The IR sensitivity of synchronous
populations of these diploid strains was examined as described above.
JPY145 strain did exhibit an increase in IR
resistance relative to its haploid counterpart JPY69 (compare Fig. 2
and Fig. 4) (7), the G2-synchronous
mre11 diploid culture did not show increased survival
relative to that of the G1-synchronous culture (Fig. 4).
Instead, we reproducibly observed a two- to threefold decrease in
survival of mre11 diploid cells in G2 (1.5% survival) relative to that of G1-synchronous cultures
(3.3% survival) (Fig. 4). This effect was not due to mating-type
hemizygosity, as MATa/MAT strains
synchronized in G1 by growth to saturation exhibited an
even more dramatic increase in survival following IR (data not shown).
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mutation partially
suppressed the IR sensitivity of the diploid mre11 strain
JPY145. Survival of the homozygous mre11 hdf1 diploid
strain JPY260 was 6.3-fold greater than that of the diploid
mre11 strain following irradiation of asynchronous
cultures (Fig. 4). mre11 hdf1 diploid cells arrested
in G1 and G2 exhibited 1.7- and 4-fold
increases in survival, respectively, relative to that of
mre11 cells.
The IR sensitivity of mre11 diploid cells suggested that
mre11 strains are defective in IR-induced interhomologue
recombination, as shown previously for rad50 strains
(33). We measured the induction of interhomologue
recombination by IR in the diploid strain JPY264 transformed with
either a wild-type MRE11 expression construct (pScM11-314)
or an empty vector. JPY264 is an Mre11-deficient MATa/mat::hisG diploid
strain heterozygous for can1 and hom3 on opposite
arms of chromosome V. In this strain, the frequency of interhomologue
recombination can be determined by scoring the frequency of
canavanine-resistant methionine prototrophs (7, 16).
Heteroallelic (intragenic) recombination cannot be distinguished from
intergenic recombination by this assay.
As in previous studies, we found that spontaneous interhomologue
recombination was increased in mre11 diploid cells
relative to that of the wild type, with frequencies of 4.0 × 103 and 3.7 × 104 recombinants per
viable cell, respectively (1). We observed greater IR
induction of interhomologue recombination in both wild-type and
mre11 G1-synchronous cells relative to that
of asynchronous and G2-synchronous cultures (Table
3), consistent with the observation that
sister chromatids are preferred over homologous chromosomes as
templates for recombinational repair (20). However, the
IR-induced frequency of interhomologue recombination in the
mre11 strain was indistinguishable from that of the wild
type at a dose of 50 Gy (Table 3).
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strain relative to that of the wild-type
(pScM11-314) transformants (Table 3), with frequencies of 4.7 × 104 and 7.3 × 104 recombinants per
viable cell, respectively. In contrast, the frequency of IR-induced
interhomologue recombination observed in G1-synchronous
mre11 cultures (31.5 × 104) was not
different from that of the wild type (35.0 × 104)
following irradiation at 150 Gy (Table 3). The extent to which IR-induced interhomologue recombination was reduced in the asynchronous and G2-synchronous mre11 cells is consistent
with the decrease in cell survival at this dose. The negative IR
induction of recombinants in the mre11 strain reflects
that the degree of cell killing at this dose exceeds the frequency of
viable recombinants.
Mre11 nuclease activity in homologous recombination. The data described above indicates that Mre11 deficiency profoundly impairs homologous recombination. Since nucleolytic processing is required for homologous recombination (15, 35), we asked whether the role of the Mre11-Rad50-Xrs2 complex in homologous recombination was dependent upon the nuclease activity of Mre11 (12, 27, 29, 36, 37). For this analysis, we used the mre11-3 mutant, in which the conserved histidine residue at position 125 is altered (7). Alteration of this residue in the Scmre11 H125N allele inactivates the nuclease function of Mre11 and disrupts the early stages of meiotic recombination (27). Like Scmre11 H125N, mre11-3 mutants are unable to produce viable spores (data not shown).
The haploid mre11 strains JPY69, JPY97, and JPY98 were
each transformed with a centromeric plasmid containing no insert (empty vector) or MRE11 or mre11-3 (both expressed from
the ADH1 promoter) (7). Transformants were
examined with respect to cell survival of synchronous cultures
following irradiation and IR-induced SCR as described above.
As with the wild-type JPY69 transformants, the presence of the sister
chromatid in the G2-synchronous mre11-3
transformants increased cell survival following IR relative to that of
G1-synchronous cultures. Asynchronous and
G2-synchronous cultures of mre11-3 transformants
exhibited 37 and 42% survival, respectively, whereas G1-synchronous cells exhibited 2% survival (Fig.
5). Consistent with cell survival, we
found that the frequency of IR-induced SCR in the haploid JPY69
mre11-3 transformants was 14.6 × 106
(Table 2), indistinguishable from that of the wild type (13.1 × 106).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We examined the response of mre11 mutants to DSBs
using cell survival, SCR, and interhomologue recombination assays. We
found that Mre11 deficiency leads to a dramatic reduction in homologous recombination, as evidenced by decreased survival following irradiation of synchronous cultures and by decreased frequencies of IR-induced sister chromatid and interhomologue recombination. Although previous studies have implicated the S. cerevisiae Mre11-Rad50-Xrs2
protein complex in NHEJ, this study clearly indicates that the primary role of the complex in the cellular DNA damage response is in facilitating homologous recombination. This role in the damage response does not appear to depend upon the nuclease activities exhibited by the complex, as a nuclease-deficient allele of
mre11 did not affect homologous recombination in our assays.
It is likely that deficiency in the S. cerevisiae
Mre11-Rad50-Xrs2 protein complex destabilizes the association of
homologous chromatids during recombinational DNA repair, thereby
impairing homologous recombination as well as NHEJ (26). In
homologous recombination, the effect is more severe for SCR than for
interhomologue recombination. For example, we observed that
G1-synchronous mre11 diploid cultures
were more IR resistant than G2-synchronous diploid cultures (Fig. 4).
The mechanistic basis for the putative reduction in chromatid
interactions in S. cerevisiae Mre11-Rad50-Xrs2 protein
complex-deficient cells is not clear. In mre11 cells, the
induction of interhomologue recombination by IR treatment is reduced in
G2-synchronous cultures relative to that of
G1-synchronous cultures (Table 3), as shown previously for
wild-type cells (10). This suggests that the bias toward the
use of sister chromatids for homologous recombination is intact in
mre11 cells. The increased survival of
G1-synchronous cultures relative to that of
G2-synchronous diploid cultures (Fig. 4) is consistent with
this interpretation. The increased sensitivity of G2
cultures suggests that the completion, rather than the initiation, of
SCR events is affected by Mre11 deficiency and that reduced survival
relative to that of G1 cultures may be caused by abortive SCR events.
The spontaneous hyperrecombination phenotype observed in
mre11, rad50, and xrs2
strains is seemingly paradoxical in light of the homologous
recombination defects in these mutant strains (1, 3, 18).
Two factors may interact to influence the frequency of spontaneous
interhomologue recombination. First, Mre11 deficiency may result in an
increased steady-state level of recombinogenic lesions and thereby lead
to an elevated frequency of interhomologue recombination. Second, the
slow resection of DSB ends in mre11 strains may lead to
shorter heteroduplex tracts during gene conversion. The result would be
an apparent increase in gene conversion between heteroalleles in a
diploid strain (14, 30). This scenario can also explain the
observation that at low doses of IR (50 Gy), both wild-type and
mre11 mutants exhibited an induction of can1
recombinants (Table 3), despite a relative decrease in survival of the
mre11 strain (data not shown). That is, although
IR-induced DNA damage is repaired inefficiently in the
mre11 strain, the decreased number of successful
recombination events that do occur do so with DNA ends that are
minimally resected and are thus more likely to result in a conversion
of the heteroallele. At higher doses of IR (150 Gy), the
frequency of cell death in mre11 cells exceeds the
frequency of successfully completed interhomologue recombination
events. Consequently, we observed a negative induction of
interhomologue recombination in mre11 cells at this dose
(Table 3).
In contrast, the spontaneous rates of SCR among null mutants of the
S. cerevisiae Mre11-Rad50-Xrs2 protein complex are
indistinguishable from each other and from rates in wild-type cells.
However, the frequency of IR-induced SCR events is reduced in
mre11 cells relative to that in the wild type (Table 2).
Although the magnitude of the observed effects is subtle, these data
offer evidence that the repair of IR-induced DNA damage may be
mechanistically distinct from the repair of DNA lesions that lead to
spontaneous SCR. In this regard, it is important to consider the
possibility that spontaneous SCR events occur in close proximity to the
replication fork to repair spontaneously occurring DSBs
(22), whereas IR-induced events occur at essentially random locations.
The homologous recombination defects we observed may not be fully explained by the hypothesized reduction in chromatid association. Rather, data presented here and elsewhere suggest that the S. cerevisiae Mre11-Rad50-Xrs2 protein complex also plays a role in facilitating end resection at DSB sites. Resection in the 5' to 3' direction to create a protruding 3' end is a requisite first step in the homologous recombination process (15, 35). A number of studies have shown that the rate of 5' to 3' end resection at HO-induced DSBs is reduced in mre11, rad50, and xrs2 deletion mutants (14). The magnitude of IR sensitivity we observed is disproportionate to the relatively modest end resection defects at HO-induced DSBs in these mutants (19, 24). However, IR-induced DSBs exhibit a significant degree of chemical and structural heterogeneity (17); therefore the end resection defects associated with Mre11 deficiency may be much more pronounced at IR- than at HO-induced DSBs.
In contrast to Mre11 deficiency, Hdf1 deficiency is associated with a
sharp increase in the rate of end resection at HO-induced DSBs. Mre11
deficiency is apparently epistatic to Hdf1 deficiency in this regard,
since resection rates are similar in mre11 and mre11 hdf1 mutants (24).
However, we observed partial suppression of mre11 IR
sensitivity in mre11 hdf1 mutants. We have
hypothesized that homologous recombination defects in
mre11 mutants result from impaired DSB end resection. We
infer that the partial suppression of IR sensitivity in
mre11 hdf1 double mutants may indicate that
Hdf1 deficiency does indeed lead to a subtle increase in the rate of
end resection at IR-induced DSBs. Given the relatively subtle degree of
suppression observed, the increased rate of end resection imparted by
Hdf1 deficiency in the mre11 background would not
necessarily have been detectable by the physical methods employed by
Lee et al. (24).
In this context, it is noteworthy that
mre11-3-expressing strains, in which Mre11 nuclease activity
is presumably abolished, are not grossly deficient in the resection of
HO-induced DSBs (23a), do not exhibit mitotic homologous
recombination defects, and do not show markedly increased IR
sensitivity (references 7 and 27 and this study).
Hence, although deficiency in the S. cerevisiae
Mre11-Rad50-Xrs2 protein complex reduces the rate of DSB end resection
in vivo, the complex may not be directly responsible for resection
activity. The complex does specify nuclease activity, although Mre11
homologues from S. cerevisiae and humans exhibit 3' to
5' rather than 5' to 3' exonuclease activity in vitro. The reduction of
5' to 3' exonuclease activity in mre11, rad50, and xrs2 mutants suggests the
possibility that the complex regulates or otherwise facilitates the
activity of a bona fide 5' to 3' exonuclease in vivo (12, 27, 29,
36, 37).
In summary, these data illustrate the central importance of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in homologous recombination. The phenotypic features of mutations affecting the complex suggest that the complex is required to establish chromatid interactions and suggest a structural, rather than enzymatic, role in the recombinational DNA repair process. The data are also compatible with a substantial role for the complex in DSB end resection to facilitate homologous recombination.
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ACKNOWLEDGMENTS |
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We are grateful to the members of our lab for insights throughout the course of this study, Michael Fasullo and Jim Haber for providing reagents, Barbara Garvik for advice and for providing yeast strains, and Mark Kaplan, Doug Bishop, and Michael Lichten for critical reading of the manuscript.
This work was supported by the Milwaukee Foundation, the Howard Hughes Medical Institute, the National Cancer Institute, NIH grant GM56888 (to J.H.J.P.), NIH predoctoral training grant 5T32GM07133 (to D.A.B.), and cancer biology postdoctoral training grant NCI T32-CA09471 to the University of Wisconsin Comprehensive Cancer Center (B.K.B.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Genetics, University of Wisconsin Medical School, 445 Henry Mall, Madison, WI 53706. Phone: (608) 265-6043. Fax: (608) 262-2976. E-mail: jpetrini{at}facstaff.wisc.edu.
Manuscript 3538 from the University of WisconsinMadison
Laboratory of Genetics.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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