From the Radiation and Genome Stability Unit, Medical Research Council, Harwell, Oxfordshire OX11 0RD, United Kingdom
Received for publication, March 16, 2001, and in revised form, April 10, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human XRCC2 gene was recently
identified by its ability to complement a hamster cell line, irs1,
which is sensitive to DNA-damaging agents and shows genetic
instability. The XRCC2 protein is highly conserved in mammalian species
and has structural features, including a putative ATP-binding domain
(P-loop), consistent with membership of the RecA/RAD51 family of
recombination-repair proteins. We show that a hybrid XRCC2-green
fluorescent protein, which was found to be functional by
complementation, localizes to the nucleus. We have established a
functional link between XRCC2 and RAD51 by looking at
damage-dependent RAD51 focus formation in the irs1 cell
line. Little or no formation of RAD51 foci occurred in irs1. This
effect was specific to the loss of XRCC2 because
transfection of the gene into irs1 restored normal levels of focus
formation. Surprisingly, XRCC2 genes carrying site-specific
mutations in P-loop residues were found to be able to complement the
XRCC2-deficient irs1 line for a number of different end points. We
conclude that XRCC2 is important in the early stages of homologous
recombination in mammalian cells to facilitate
RAD51-dependent recombination repair but that it
does not make use of ATP binding to promote this function.
The repair of DNA damage by homologous recombination has an
important function in maintaining genetic stability in cells. In
bacteria, the RecA protein has a central role in the recombination process, and in the last decade RecA-like proteins have been discovered in eukaryotes. In particular the RAD51 protein is highly conserved from
yeast to humans and has been shown to have similar attributes to RecA
(1, 2). Mutations in both RecA and RAD51 cause severe defects in
recombination and extreme sensitivity to DNA-damaging agents. RecA acts
directly in recombination processes in which, in the presence of ATP,
it forms a polymer on single-stranded DNA and promotes strand exchange
with a homologous sequence (3). Using molecular recombination assays,
yeast (Saccharomyces cerevisiae) and human RAD51 proteins
have been shown to promote strand exchange similarly, although some of
the biochemical properties of RAD51 differ from those of RecA. For
example, purified RecA preferentially binds to single-stranded DNA and
hydrolyzes ATP at a relatively high rate, whereas the yeast and human
RAD51 proteins bind equally to single- and double-stranded DNA and show
a much lower rate of ATP hydrolysis (1). All members of this family
have a highly conserved sequence motif, first described by Walker
et al. (4), that has been linked to ATP binding. The
flexible loop of this motif (Walker box A) interacts with the
phosphates of ATP and is therefore sometimes called the P-loop.
In S. cerevisiae, two further members of the
RecA/RAD51family of proteins facilitate homologous recombination in
mitotic cells; Rad55p and Rad57p form a heterodimer and stimulate
RAD51-mediated recombination (5). Yeast mutants that lack these
recombination proteins are also extremely sensitive to agents causing
severe forms of damage to DNA, such as double strand breaks and
interstrand cross-links (6). These additional recombination proteins
also have homologues in somatic mammalian cells, in which five
RAD51-like proteins have recently been discovered (XRCC2, XRCC3,
RAD51L1, RAD51L2, and RAD51L3) (7).
The XRCC2 gene was identified and cloned by its ability to
complement the damage-sensitive phenotype of the irs1 hamster cell line
(8-10). The irs1 line, isolated in this laboratory several years ago
(11), was found to be sensitive to a variety of agents including
ionizing radiation, ultraviolet light, alkylating agents, and
especially DNA cross-linking agents such as mitomycin-C. It also shows
spontaneous genetic instability with increased frequencies of mutations
(12), chromosomal aberrations (13), and chromosome nondisjunction (14).
It has recently been shown that the repair of a site-specific double
strand break by recombination is severely reduced in irs1 compared with
the paternal V79 cells (15). The results of two-hybrid interactions
involving XRCC2 have led to the speculation of an indirect
association with RAD51 through other recombination proteins (16).
However, as yet there is no direct evidence for the involvement of
XRCC2 in RAD51-dependent recombination processes.
It has been shown that RAD51 can be detected in discrete nuclear foci
following DNA damage to mammalian cells (17-19). In the present study,
we considered the localization of XRCC2 in mammalian cells and
established a functional link between XRCC2 and RAD51 by looking at
damage-dependent RAD51 focus formation in the
XRCC2-deficient cell line irs1. Additionally, we have created a number
of mutations of XRCC2 in the Walker A motif to determine the
importance of this site in the functioning of XRCC2.
Mammalian Cell and Bacterial Culture Methods--
Wild-type V79
and XRCC2-deficient irs1 cells were grown as monolayers at 37 °C in
minimal essential medium (Life Technologies, Inc.) supplemented with
10% fetal calf serum and antibiotics. To assay the sensitivity of
individual clones to the DNA cross-linking agent mitomycin-C
(MMC)1, cells were respread
into 24-well plates at 100-300 cells/well containing graded
concentrations of MMC (0-100 nM). After 10-12 days of
incubation, the density of cell growth was assessed visually (see Table
I). Bacterial cultures were grown in Luria-Bertani medium supplemented
with appropriate antibiotics; plasmids were propagated in strain
DH5 Expression of XRCC2-GFP Protein in irs1
Cells--
XRCC2 was amplified from a human cDNA
library using Pfu polymerase (Stratagene) with primers to
incorporate restriction sites for cloning into pEGFP-N1
(CLONTECH) to create an in-frame fusion of the
EGFP gene to the 3'-end of XRCC2. Approximately 4 µg of plasmid DNA was transfected into irs1 cells by electroporation (Bio-Rad Gene Pulser at 400 V/500 microfarads) in Cytomix buffer (20).
Cells were selected for 14 days in 500 µg/ml G418 (Life Technologies,
Inc.). G418-resistant clones were tested for MMC resistance using
24-well plates (see above) to demonstrate XRCC2 function. MMC-resistant
cells were grown on glass coverslips, and the fusion protein was
visualized at 488 nm using a Bio-Rad MRC600 laser scanning confocal microscope.
Site-directed Mutagenesis--
Human XRCC2 cDNA
was cloned into pBS as a template for site-directed mutagenesis.
Mutagenic polymerase chain reaction was carried out using the
QuikChange site-directed mutagenesis protocol (Stratagene) with primers
designed to create changes at the highly conserved lysine 54 residue
(K54A, K54R, and Clonogenic Survival Following MMC and X-ray Treatment--
Pools
of ~100 G418-resistant clones carrying mutated plasmids were grown in
medium containing 100 µg/ml G418 and MMC (0-100 nM) for
10-12 days. Similarly, pooled G418-resistant cells were irradiated
with either 0-, 2.5-, 5-, or 10-Gy x-rays and then grown in medium
containing 100 µg/ml G418. After 10-12 days, colonies were stained
with methylene blue and counted.
Cytogenetic Analysis--
Metaphases were collected from cells
grown in flasks by exposure to colcemid (0.05 µg/ml) for 2 h.
Cells were trypsinized and incubated in hypotonic solution (1:1; 0.075 M KCl, 0.034 M trisodium citrate) for 8 min at
37 °C. Cells were fixed in 3:1 ethanol:acetic acid and dropped onto
slides before staining with Giemsa for analysis. Approximately 100 metaphases/cell type were analyzed for both chromatid and chromosome aberrations.
RAD51 Immunofluorescence--
Cells were grown on glass
coverslips to subconfluent levels and irradiated with 10-Gy x-rays.
After 0-5 h of incubation at 37 °C, cells were fixed for 2 h
at 4 °C in 1% paraformaldehyde in PBS. Fixed cells were
permeabilized for 10 min at room temperature in 0.1% Triton
X-100/0.1× SSC and then blocked for 1 h at room temperature in
PBS/5% normal horse serum. Rabbit anti-hRAD51 antibody (FBE1, a
generous gift from S. C. West, Imperial Cancer Research Fund Clare Hall Laboratories) was applied at a dilution of 1:100 in
PBS/5% horse serum and incubated overnight at 4 °C. Cells were treated with anti-rabbit IgG/Cy5 (Jackson ImmunoResearch) at 1:100 dilution for 1 h at room temperature in the dark, and then the coverslips were mounted in Vectashield anti-fade medium. Cells were
analyzed by confocal microscopy at 633 nm, and images were selected at
random. Immunofluorescence images were scored blindly by two
independent scorers who recorded the number of discrete strongly
fluorescing nuclear foci present in each cell. Approximately 50 cells
were counted per data point, and data were compared statistically to
assess homogeneity using the Mann-Whitney U test. One scorer consistently scored about 30% more foci than the other, but identical trends of increased focus formation with irradiation and with time
after irradiation (of cell lines with wild-type XRCC2) were found for each scorer.
Cell Cycle Analysis--
Cells were seeded at 5 × 105/75-cm2 flask at 16 h before
x-irradiation. Cells were then harvested at intervals of up to 7 h, washed three times with PBS, and fixed in 70% ethanol at 4 °C for 30 min. Fixed cells were incubated for 60 min at 37 °C with 0.5% RNaseA (Sigma) before the addition of propidium iodide to 50 µg/ml final concentration. Flow cytometric analysis (>10,000 cells/sample) was performed on a FACSort (Becton Dickinson).
Western Blotting--
Cells were grown to subconfluent levels in
tissue culture flasks, irradiated with 10-Gy x-rays, and harvested
after a further 5 h growth. Cells were resuspended in distilled
water and heated for 15 min at 95 °C, and total protein was measured
using the Bradford assay (Bio-Rad). The lysate was resuspended in an
equal volume of 2× Laemmli/glycerol buffer and treated for a further 5 min at 95 °C. A 20-µg sample was separated on a 7.5%
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membrane (Millipore). RAD51 was detected using the FBE1 antibody at a
dilution of 1:2500 in PBS, 0.1% Tween 20, 5% bovine serum albumin,
5% nonfat dried milk. Anti-rabbit IgG/horseradish peroxidase conjugate
was used as the secondary antibody (Promega) at a dilution of 1:5000,
and bands were visualized using ECL (Perkin Elmer Life Sciences
Renaissance) according to the manufacturer's protocol) and Biomax film
(Eastman Kodak Co.). Anti- XRCC2 Is Localized in the Nucleus of Mammalian Cells--
The
human XRCC2 protein was expressed as a fusion to the N terminus of the
green fluorescent protein (GFP) in XRCC2-defective irs1 hamster cells.
This construct was tested for functional ability by transfection into
the irs1 line and exposure to a range of concentrations of the DNA
cross-linking agent MMC. As shown previously (9), the expression of the
wild-type human XRCC2 gene in irs1 cells gives a
good, although not complete, level of complementation of MMC
sensitivity (Table I). In the same test,
the XRCC2-GFP hybrid gene had no loss of complementing
ability when compared with the XRCC2 cDNA (Table I).
Although the expression of GFP alone occurred throughout the cell (data
not shown), its expression was primarily nuclear when associated with
XRCC2 (Fig. 1).
XRCC2 Is Required for the Formation of RAD51 Foci in Response to
DNA Damage--
To assess the function of XRCC2 specifically in
recombination-repair processes, we measured the formation of
radiation-induced RAD51 foci in wild-type (V79) and irs1 hamster cells.
Cells were irradiated with 10-Gy x-rays, and the numbers of foci were
counted at hourly intervals for up to 5 h. In agreement with
previous studies (17, 21, 22), a low level of focus formation was seen
in unirradiated cells and in cells fixed immediately after irradiation
(generally <2 foci/cell; see Fig.
2A). In V79 cells the numbers
of foci increased significantly following x-irradiation (Fig.
2A). Although there was some fluctuation in the numbers of
foci seen in V79 cells with time following irradiation (Fig. 2B), this variation was not significant over 1-5 h after
irradiation (p = 0.8). In the XRCC2-deficient irs1
cells, little focus formation was found (Fig. 2A), although
there is some evidence for a slow accumulation of foci with time.
However, this level is on average about 5-fold lower (p = 10
It has been shown that RAD51 foci in unirradiated cells form primarily
in S-phase (21), raising the question of whether differences in cell
cycle distribution may influence the ability to detect foci in irs1
after irradiation. However, cell cycle profiles for irradiated V79 and
irs1 cells were very similar with most cells blocked in G2
phase at 5 h after a dose of 10 Gy (Fig. 3A). It is also possible that
the formation of RAD51 foci is affected by the levels of RAD51 protein
in irs1 relative to V79, but we found that RAD51 levels were similar in
the two cell lines, both before and after irradiation (Fig.
3B).
Transfection with the Human XRCC2 Gene Restores RAD51 Focus
Formation--
The stable transfection of the irs1 cells with human
XRCC2 genomic (P1-artificial chromosome) DNA (8) or cDNA
(Table I) gave clonal lines with a good level of correction of
DNA-damage sensitivity. These transfected lines were also found to have
a restored ability to form RAD51 foci after irradiation (Fig.
2A), whereas RAD51 protein levels were similar to those in
irs1 and V79 (Fig. 3B). This finding implicates the
XRCC2 gene specifically in correct focus formation. The time
course for the development of foci was apparently slower in the
XRCC2-transfected lines than for V79, possibly because the
human gene is not as efficient in a hamster cell background (Fig.
2B).
Site-directed Mutations of the Walker Box A of XRCC2 Have Little
Effect on Complementation of Mitomycin-C Resistance--
To gain
further information on the function of XRCC2, we mutated the highly
conserved Walker box A (putative ATP binding) motif in several
different ways. It has been shown previously that the invariant lysine
residue of this motif is critical to the function of RecA or RAD51
proteins (see "Discussion"). Initially therefore we mutated this
residue to either a conservative (K54R) or nonconservative (K54A)
alternative in XRCC2 as well as deriving a mutant form in which the
lysine was deleted along with an adjacent glycine ( The Influence of Walker Box A Mutants on X-ray Survival,
Chromosomal Aberrations, and RAD51 Focus Formation--
To
generalize our findings with MMC sensitivity, we tested some of the
mutant XRCC2 genes for other responses. As expected the differential between wild-type and mutant XRCC2 genes
was much less for x-rays than for MMC treatment (11), and in this case
the
The loss of XRCC2 seriously influences genetic stability even when
XRCC2-deficient cells are not exposed to exogenous DNA-damaging agents.
We have shown previously that the spontaneous chromosome aberration
frequency is high in irs1 cells but is restored to near normal levels
in cells transfected with the XRCC2 cDNA (9). Consistent
with the survival data, examination of the chromosomes of irs1 cells
transfected with the different mutant forms of XRCC2 showed that the
alteration or loss of the conserved glycine and lysine residues of the
P-loop did not affect the ability of the gene to confer genetic
stability (Table II).
Repeat experiments assessing the damage-dependent RAD51
focus formation are shown in Fig. 6. In
this assay, as was found for the mitomycin-C response, the K54A
mutation does not show a significant difference from the wild-type
cDNA, and the We have found that the XRCC2 protein is located in the nucleus,
which is consistent with a role in DNA metabolism. To test for the
involvement of XRCC2 in repair by homologous recombination, as would be
predicted from sequence homologies (7), we measured the requirement for
XRCC2 in damage-dependent RAD51-focus formation. RAD51 has
been shown to be expressed in a cell cycle-dependent fashion with the majority of expression in S- and G2-phase
cells (23-26). In previous studies it has been established that some nuclear RAD51 foci will form in the S-phase of untreated somatic cells
(21). However, the numbers of foci are substantially increased by
treatment with DNA-damaging agents, including ionizing radiation, in a
time- and dose-dependent manner (17, 19). Although
experiments shielding some cells from irradiation (27) have suggested
that RAD51 protein does not co-localize with sites of radiation damage, more recent studies using microbeams show that focus formation is
spatially related to sites of damage in the nucleus of somatic cells
(28). Additionally, human RAD51 shows co-localization to sites of DNA
damage as represented by single-stranded DNA (29) and histone H2AX
phosphorylation (30). V79 hamster cells showed extensive focus
formation within 1 h of irradiation, and thereafter the number of
foci/cell remained approximately constant. Very little RAD51 focus
formation occurred in the XRCC2-deficient irs1 cells, although there
was some evidence for a small increase with time. This small increase
could be attributable to the accumulation of cells in S- and
G2 phases of the cell cycle (Fig. 3A) because RAD51 is expressed primarily in these phases. In the irs1 cells, the XRCC2 transcript is shortened because of the loss of exon 2,2 and this will lead to a
frameshift in the sequence, giving a predicted protein of only 33 amino
acids (of which 13 are correct, compared with the full-length sequence
of 278 amino acids). Therefore, this truncated XRCC2 protein is
unlikely to have any normal activity. Additionally, we have shown that
the loss of RAD51 focus formation in irs1 is specific to XRCC2 because
the ectopic expression of full-length human XRCC2 restored
normal levels of foci following irradiation.
The XRCC2 protein has few identifiable motifs to give clues on function
except for the proposed ATP binding boxes. As noted in the
Introduction, the conserved Walker A box (or P-loop) is characteristic
of all members of the RecA/RAD51 family. However, we found that the
substitution of highly conserved residues in the P-loop has little
effect on the ability of XRCC2 to complement a number of end points
when introduced into the irs1 line, including survival in
response to MMC or x-rays and genetic instability. Even in the case of
the It seems likely therefore that in both yeast and humans, only one of
the partners in RAD51-like protein heterodimers retains the ATP binding
requirement. In recent studies there are a number of precedents for
this type of "functional asymmetry" in protein complexes that
affect repair and recombination processes. For example, the
ATP-dependent deoxyribonuclease complex in Bacillus subtilis possesses two ATP binding sequences. The mutation of the
lysine residue in the AddA subunit drastically affects function, whereas the identical mutation in the AddB subunit has only marginal effects (38). Similarly there is evidence that the partner proteins of
heterodimers involved in DNA mismatch repair, such as MSH2 and MSH6,
make different contributions to ATPase activity (39). The equivalent
mismatch repair protein in E. coli, MutS, forms homodimers,
but even in this case there is evidence from recent crystal structure
analysis that the partners do not participate equally in the ATP
binding reaction (40). This asymmetry may promote conformational
changes that assist further reactions such as DNA binding. It will be
interesting to see whether other mammalian RAD51-like proteins, some of
which are proposed to interact as dimers or in higher-order complexes
(16), have a similar functional divergence.
Our results with XRCC2 support the evolutionary conservation of
function in RAD51-like proteins from yeast to man. For meiotic recombination in yeast, Rad55p and Rad57p are required for RAD51 focus
formation (41), and purified Rad55p/Rad57p heterodimer has been shown
to facilitate the loading of RAD51 onto single-stranded DNA coated with
replication protein A (5). The XRCC2/RAD51L3 heterodimer in mammalian
cells may act in a similar manner to Rad55p/Rad57p. However, the
process is likely to be more complex in mammalian cells, which
include several additional RAD51-like proteins (7) that interact to
form heterodimers and possibly larger complexes (5, 16, 37). Some of
these proteins have also been shown to be required for RAD51 focus
formation, namely XRCC3 in mammalian cells (19) and RAD51L1 in chick
cells (42). Despite their apparent facilitating function, it is clear
that the RAD51-like proteins have an important part to play in
mammalian development because in the case of XRCC2 (43),
RAD51L1 (44), and RAD51L3 (45) gene disruption in
mice leads to embryonic lethality. Further, there are parallels between
XRCC2 deficiency and the disruption of the breast cancer susceptibility
genes BRCA1 and BRCA2. These genes have recently
been shown to be required for damage-dependent RAD51 focus
formation (46, 47) and have reduced levels of homologous recombination
(48-50). The BRCA genes are also required for correct
chromosome segregation at mitosis (51, 52), and we have shown that
XRCC2 and XRCC3 promote the fidelity of
chromosome segregation (14). These similarities suggest a role for
RAD51-dependent homologous recombination repair and for
XRCC2 in particular in maintaining genetic stability in mammalian cells
and perhaps in influencing the incidence of cancer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Plasmid DNA was prepared using a Qiaprep kit (Qiagen);
sequencing reactions were performed using a BigDye kit
(PerkinElmer Life Sciences).
G53K54). In addition to these mutations, a
cDNA with a fortuitous stop codon in the same region was derived
from the mutagenic polymerase chain reaction, and this was used as a
negative control. Inserts carrying mutations were subcloned into the
NheI and BamHI sites of the pIRESneo2 vector
(CLONTECH). Plasmid DNA was transfected into irs1
cells (as above), and cells were selected for 14 days in 500 µg/ml
G418. Six separate clones for each construct were picked and tested for
MMC resistance using the 24-well plate assay (see above). The internal
ribosome entry site vector was used to select against integration
events that disrupted the XRCC2 gene. In control
experiments, ~85% of G418-resistant clones also expressed the
XRCC2 construct (data not shown).
-tubulin antibody (1:2000) (Sigma) was
used as loading control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Growth of wild-type V79, XRCC2-deficient irs1, and transfected
derivatives of irs1 in the presence of mitomycin-C
, no growth.
View larger version (125K):
[in a new window]
Fig. 1.
Nuclear localization of
XRCC2-GFP protein. Scale bar, 50 µm).
6) than that seen for the XRCC2-proficient V79 cells
(Fig. 2B).
View larger version (51K):
[in a new window]
Fig. 2.
Damage-dependent RAD51 focus
formation. A, foci in wild-type V79, irs1, and irs1
cells transfected with XRCC2 with and without x-ray exposure
(10 Gy/5 h). Representative fields are shown. B, time course
of focus formation following 10-Gy x-rays. Results from representative
experiments are shown. PAC, P1-artificial chromosome.
View larger version (30K):
[in a new window]
Fig. 3.
A, cell cycle distributions of V79 and
irs1 cells in response to 10-Gy x-rays. B, Western blot of
RAD51 in V79, irs 1, and irs1 cells transfected with XRCC2
with and without 10-Gy irradiation. In a representative experiment
using the -tubulin response as a loading control, the RAD51 signal
strength showed no significant differences between cell lines or before
and after irradiation.
G53K54) (Fig.
4). Negative controls in this experiment were provided by a mutation leading to a stop codon in this region of
the XRCC2 sequence and by the cloning vector alone. Each mutant gene
was verified by sequencing and was then transfected into irs1 cells to
assess the functional complementation. We initially used MMC survival
to test the response of the mutated genes because XRCC2-deficient cells
show an extreme sensitivity to this DNA-damaging agent. The ability to
complement MMC sensitivity was first tested in the simple growth assay
from a standard cell inoculum in 24-well plates (see "Experimental
Procedures"). As seen in Table I, each of the mutants tested apart
from the truncation showed little or no reduction in the ability to
complement the MMC sensitivity of irs1 relative to the wild-type
cDNA. To check that there was no loss of complementing ability by
more stringent procedures, allowing for the cloning efficiency of each
transfected cell population, multipoint survival curves were carried
out with pooled clones for several of the mutants (Fig.
5A). It is seen that the K54A mutant shows little or no loss of complementing ability compared with
the wild-type cDNA, whereas the
G53K54 mutant has a small but
consistent reduction in this capacity.
View larger version (17K):
[in a new window]
Fig. 4.
Amino acid sequence of part of the human
XRCC2 gene including the Walker box A, showing the
locations of site-directed mutants. Those locations are
shown in italics or by missing residues.
View larger version (14K):
[in a new window]
Fig. 5.
Survival curves for V79, irs1, and irs1 cells
transfected with different wild-type and mutant XRCC2
cDNAs. A, mitomycin-C (mean and range of 2-3
experiments). B, x-rays (single experiments). wt,
wild type.
G53K54 mutant showed no significant difference in sensitivity from the wild-type cDNA (Fig. 5B).
Frequencies of chromosomal aberrations in wild type V79,
XRCC2-deficient irs1, and transfected derivatives of irs1
G53K54 mutation shows only a small reduction in
focus formation.
View larger version (31K):
[in a new window]
Fig. 6.
RAD51 focus formation in V79, irs1, and irs1
cells transfected with XRCC2 cDNA (wild-type or
mutant forms). Means and standard error of data from 2-5
experiments for unirradiated and irradiated (10 Gy/5 h) cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G53K54 mutation, which deletes two key residues within the
Walker box A motif, there was only a small negative effect relative to
wild-type cDNA when measured by mitomycin-C response (Fig.
5A) and focus formation (Fig. 6). We especially targeted the
lysine residue in our XRCC2 mutations because this site is conserved in
all NTP-binding proteins with the P-loop motif (31). In ATPases the
conserved lysine is probably important to the conformation of the
P-loop and for direct interaction with the
- and
-phosphates of
the bound ATP. Consistent with this degree of conservation, the
mutation of this residue in ATP-binding proteins generally compromises
function. This is certainly true for Escherichia coli RecA
for which even the relatively conservative K72R mutation gives a null
phenotype (32) although the mutant protein retains the ability to
promote homologous pairing of DNA strands (33). S. cerevisiae Rad51p altered at this P-loop lysine similarly has a
compromised function; the K191A mutation gives a null phenotype,
whereas K191R has a partially defective phenotype (34). In human RAD51,
again a conservative mutation of this lysine residue (K133R) gives a
protein capable of partially restoring function in chick
RAD51-defective cells, whereas the nonconservative K133A mutation
cannot restore function (35). Therefore, our finding that such
mutations do not lead to a compromise of XRCC2 function is perhaps
surprising, although not without precedent. Mutation of the P-loop
lysine in the S. cerevisiae RAD51-like protein Rad57p, even
to a nonconservative change (K133A), does not affect its function in
DNA recombination repair. In contrast Rad55p, the yeast RAD51-like
protein, which forms a heterodimer with Rad57p, does require an intact
lysine residue in the P-loop motif for function (36). We have recently
shown that, similarly to Rad55p/Rad57p, XRCC2 forms a heterodimer with
another human RAD51-like protein, RAD51L3, and that RAD51L3 has
significant DNA-stimulated ATPase activity (37).
![]() |
ACKNOWLEDGEMENTS |
---|
We are very grateful to Steve West for supplying the RAD51 antibody and to Carol Griffin for help with cytogenetic analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by Contract FIGH-CT1999-10010 from the European Commission.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.
Recipient of a studentship from the Medical Research Council.
§ To whom correspondence should be addressed: Medical Research Council, Radiation & Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, UK. Tel.: 44-1235-834393; Fax: 44-1235-834776; E-mail: j.thacker@har.mrc.ac.uk.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M102396200
2 C. Tambini and J. Thacker, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MMC, mitomycin-C; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Gy, gray.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baumann, P., and West, S. C. (1998) Trends Biochem. Sci. 23, 247-251[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bianco, P. R., Tracy, R. B., and Kowalczykowski, S. C. (1998) Front. Biosci. 3, D570-D603[Medline] [Order article via Infotrieve] |
3. | Cox, M. M. (1999) Prog. Nucleic Acid Res. Mol. Biol. 63, 311-366[Medline] [Order article via Infotrieve] |
4. | Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951[Medline] [Order article via Infotrieve] |
5. | Sung, P. (1997) Genes Dev. 11, 1111-1121[Abstract] |
6. | Game, J. C. (1993) Semin. Cancer Biol. 4, 73-83[Medline] [Order article via Infotrieve] |
7. | Thacker, J. (1999) Trends Genet. 15, 166-168[CrossRef][Medline] [Order article via Infotrieve] |
8. | Tambini, C. E., George, A. M., Rommens, J. M., Tsui, L. C., Scherer, S. W., and Thacker, J. (1997) Genomics 41, 84-92[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Cartwright, R.,
Tambini, C. E.,
Simpson, P. J.,
and Thacker, J.
(1998)
Nucleic Acids Res.
26,
3084-3089 |
10. | Liu, N., Lamerdin, J. E., Tebbs, R. S., Schild, D., Tucker, J. D., Shen, M. R., Brookman, K. W., Siciliano, M. J., Walter, C. A., Fan, W., Narayana, L. S., Zhou, Z. Q., Adamson, A. W., Sorensen, K. J., Chen, D. J., Jones, N. J., and Thompson, L. H. (1998) Mol. Cell 1, 783-793[Medline] [Order article via Infotrieve] |
11. | Jones, N. J., Cox, R., and Thacker, J. (1987) Mutat. Res. 183, 279-286[Medline] [Order article via Infotrieve] |
12. | Thacker, J., Ganesh, A. N., Stretch, A., Benjamin, D. M., Zahalsky, A. J., and Hendrickson, E. A. (1994) Mutagenesis 9, 163-168[Abstract] |
13. | Tucker, J. D., Jones, N. J., Allen, N. A., Minkler, J. L., Thompson, L. H., and Carrano, A. V. (1991) Mutat. Res. 254, 143-152[Medline] [Order article via Infotrieve] |
14. | Griffin, C. S., Simpson, P. J., Wilson, C. R., and Thacker, J. (2000) Nat. Cell Biol. 2, 757-761[CrossRef][Medline] [Order article via Infotrieve] |
15. | Johnson, R. D., Liu, N., and Jasin, M. (1999) Nature 401, 397-399[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Schild, D.,
Lio, Y.,
Collins, D. W.,
Tsomondo, T.,
and Chen, D. J.
(2000)
J. Biol. Chem.
275,
16443-16449 |
17. | Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., and Ward, D. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2298-2302[Abstract] |
18. | Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435[Medline] [Order article via Infotrieve] |
19. |
Bishop, D. K.,
Ear, U.,
Bhattacharyya, A.,
Calderone, C.,
Beckett, M.,
Weichselbaum, R. R.,
and Shinohara, A.
(1998)
J. Biol. Chem.
273,
21482-21488 |
20. | van den Hoff, M. J., Moorman, A. F., and Lamers, W. H. (1992) Nucleic Acids Res. 20, 2902[Medline] [Order article via Infotrieve] |
21. | Tashiro, S., Kotomura, N., Shinohara, A., Tanaka, K., Ueda, K., and Kamada, N. (1996) Oncogene 12, 2165-2170[Medline] [Order article via Infotrieve] |
22. | Li, M. J., and Maizels, N. (1997) Exp. Cell Res. 237, 93-100[CrossRef][Medline] [Order article via Infotrieve] |
23. | Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D. M. (1997) Cell 88, 265-275[Medline] [Order article via Infotrieve] |
24. | Chen, F., Nastasi, A., Shen, Z., Brenneman, M., Crissman, H., and Chen, D. J. (1997) Mutat. Res. 384, 205-211[Medline] [Order article via Infotrieve] |
25. | Flygare, J., Benson, F., and Hellgren, D. (1996) Biochim. Biophys. Acta 1312, 231-236[Medline] [Order article via Infotrieve] |
26. | Yamamoto, A., Taki, T., Yagi, H., Habu, T., Yoshida, K., Yoshimura, Y., Yamamoto, K., Matsushiro, A., Nishimune, Y., and Morita, T. (1996) Mol. Gen. Genet. 251, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Nelms, B. E.,
Maser, R. S.,
MacKay, J. F.,
Lagally, M. G.,
and Petrini, J. H.
(1998)
Science
280,
590-592 |
28. |
Tashiro, S.,
Walter, J.,
Shinohara, A.,
Kamada, N.,
and Cremer, T.
(2000)
J. Cell Biol.
150,
283-291 |
29. |
Raderschall, E.,
Golub, E. I.,
and Haaf, T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1921-1926 |
30. | Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., and Bonner, W. M. (2000) Curr. Biol. 10, 886-895[CrossRef][Medline] [Order article via Infotrieve] |
31. | Saraste, M., Sibbald, P. R., and Wittinghofer, A. (1990) Trends Biochem. Sci. 15, 430-434[CrossRef][Medline] [Order article via Infotrieve] |
32. | Logan, K. M., and Knight, K. L. (1993) J. Mol. Biol. 232, 1048-1059[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Rehrauer, W. M.,
and Kowalczykowski, S. C.
(1993)
J. Biol. Chem.
268,
1292-1297 |
34. | Shinohara, A., Ogawa, H., and Ogawa, T. (1992) Cell 69, 457-470[Medline] [Order article via Infotrieve] |
35. |
Morrison, C.,
Shinohara, A.,
Sonoda, E.,
Yamaguchi-Iwai, Y.,
Takata, M.,
Weichselbaum, R. R.,
and Takeda, S.
(1999)
Mol. Cell. Biol.
19,
6891-6897 |
36. | Johnson, R. D., and Symington, L. S. (1995) Mol. Cell. Biol. 15, 4843-4850[Abstract] |
37. | Braybrooke, J. P., Spink, K. G., Thacker, J., and Hickson, I. D. (2000) J. Biol. Chem. 37, 29100-29106[CrossRef] |
38. | Haijema, B. J., Noback, M., Hesseling, A., Kooistra, J., Venema, G., and Meima, R. (1996) Mol. Microbiol. 21, 989-999[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Iaccarino, I.,
Marra, G.,
Palombo, F.,
and Jiricny, J.
(1998)
EMBO J.
17,
2677-2686 |
40. | Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Gasior, S. L.,
Wong, A. K.,
Kora, Y.,
Shinohara, A.,
and Bishop, D. K.
(1998)
Genes Dev.
12,
2208-2221 |
42. |
Takata, M.,
Sasaki, M. S.,
Sonoda, E.,
Fukushima, T.,
Morrison, C.,
Albala, J. S.,
Swagemakers, S. M.,
Kanaar, R.,
Thompson, L. H.,
and Takeda, S.
(2000)
Mol. Cell. Biol.
20,
6476-6482 |
43. |
Deans, B.,
Griffin, C. S.,
Maconochie, M.,
and Thacker, J.
(2000)
EMBO J.
19,
6675-6685 |
44. |
Shu, Z.,
Smith, S.,
Wang, L.,
Rice, M. C.,
and Kmiec, E. B.
(1999)
Mol. Cell. Biol.
19,
8686-8693 |
45. | Pittman, D. L., and Schimenti, J. C. (2000) Genesis 26, 167-173[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Yuan, S. S. F.,
Lee, S. Y.,
Chen, G.,
Song, M. H.,
Tomlinson, G. E.,
and Lee, E.
(1999)
Cancer Res.
59,
3547-3551 |
47. |
Bhattacharyya, A.,
Ear, U. S.,
Koller, B. H.,
Weichselbaum, R. R.,
and Bishop, D. K.
(2000)
J. Biol. Chem.
275,
23899-23903 |
48. | Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518[Medline] [Order article via Infotrieve] |
49. | Moynahan, M. E., Pierce, A. J., and Jasin, M. (2001) Mol. Cell 7, 263-272[Medline] [Order article via Infotrieve] |
50. | Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L., and Koller, B. H. (1999) Oncogene 18, 7900-7907[CrossRef][Medline] [Order article via Infotrieve] |
51. | Tutt, A., Gabriel, A., Bertwistle, D., Connor, F., Paterson, H., Peacock, J., Ross, G., and Ashworth, A. (1999) Curr. Biol. 9, 1107-1110[CrossRef][Medline] [Order article via Infotrieve] |
52. | Xu, X., Weaver, Z., Linke, S. P., Li, C., Gotay, J., Wang, X. W., Harris, C. C., Ried, T., and Deng, C. X. (1999) Mol. Cell 3, 389-395[Medline] [Order article via Infotrieve] |