Department of Molecular Biology and Skaggs
Institute for Chemical Biology, The Scripps Research Institute, La
Jolla, California 920371; Life Sciences
Division, Lawrence Berkeley National Laboratory, Berkeley, California
947202; and Radiation Oncology Research
Laboratory, Department of Radiation Oncology, and The Marlene and
Stewart Greenebaum Cancer Center, University of Maryland School of
Medicine, Baltimore, Maryland 212013
The processing of DNA double-strand breaks is a critical event in
nucleic acid metabolism. This is evidenced by the severity of
phenotypes associated with deficiencies in this process in multiple
organisms. The core component involved in double-strand break repair in
eukaryotic cells is the Mre11-Rad50 protein complex, which includes a
third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11
and Rad50 have been identified in all kingdoms of life, while the Nbs1
protein family is found only in eukaryotes. In eukaryotes the
Mre11-Rad50 complex has nuclease activity that is modulated by the
addition of ATP. We have isolated the Mre11 and Rad50 homologues from
the thermophilic archaeon Pyrococcus furiosus and
demonstrate that the two proteins exist in a large, heat-stable complex
that possesses single-strand endonuclease activity and ATP-dependent
double-strand-specific exonuclease activity. These findings verify the
identification of the P. furiosus Rad50 and Mre11
homologues and demonstrate that functional homologues with similar
biochemical properties exist in all kingdoms of life.
 |
INTRODUCTION |
The ability to properly repair DNA
double-strand breaks (DSBs) is of paramount importance for cellular
survival and maintenance of genomic stability. These breaks can be
caused by ionizing radiation or genotoxic chemicals, or they can occur
spontaneously during DNA synthesis or as part of a number of programmed
cellular event such as mating type switching and meiotic crossing over
in Saccharomyces cerevisiae and V(D)J recombination in the
mammalian immune system (8, 12, 24). The importance of the
repair pathways involved in processing DSBs has been demonstrated by
the severe phenotypes observed in a number of organisms associated with
the inability to repair this type of DNA damage (7, 17). The
genes of the S. cerevisiae RAD52 epistasis group
(RAD50 to 59, MRE11, XRS2, and RPA) have been determined to be the main effectors of
DSB repair in this organism (7). The understanding of the
functions of the gene products of this epistasis group has led to a
greater understanding of DSB repair in multiple organisms, given that most of the members of the RAD52 epistasis group are
conserved in a number of species including mammals (17).
Among the members of the RAD52 epistasis group,
MRE11, RAD50, and XRS2 have been shown
to function as a stable multiprotein complex that possesses nuclease
activity (Mre11) and ATP-dependent DNA binding activity (Rad50)
(6, 13, 18). In humans, the homologues of the
MRE11 and RAD50 gene products have been
previously isolated and found to be members of a multiprotein complex
that possesses nuclease activity, DNA binding activity, and DNA
unwinding activity (15, 16, 22). A third member of the human
complex, p95, has been isolated and found to be the protein that is
defective in the chromosomal instability disorder Nijmegen breakage
syndrome (NBS) (3, 23). Furthermore, mutations in the
hMRE11 gene lead to a disease similar to ataxia
telangiectasia termed ataxia telangiectasia-like disorder (ATLD)
(21). Patients with ATLD are similar to those with NBS in
that they display an abnormal response to the induction of DNA DSBs.
Genetic studies in S. cerevisiae have implicated the complex
in the repair of DSBs by both the homologous recombination repair
pathway and the nonhomologous end-joining pathway (9).
Examination of the homologues of Mre11, Rad50, and p95 by sequence
database survey has shown that Rad50 and Mre11 are conserved in all
kingdoms of life, while functional homologues of Nbs1 exist only in
eukaryotes (1). Furthermore, purification of the
Escherichia coli Mre11 and Rad50 homologues SbcD and SbcC
shows that there do not appear to be any additional factors associated
with these proteins (4). Thus, it appears that Mre11 and
Rad50 comprise the core enzymatic members of this conserved
multiprotein machine. To date there has been no characterization of
this evolutionarily conserved complex from an archaeon. While Mre11
homologues are readily observed in archaea, the identity of an archaeal
Rad50 is unclear because the protein is a member of the structural
maintenance of chromosomes (SMC) family of proteins, of which there are
several homologues in a given organism.
We report here the isolation and initial characterization of the
homologues of MRE11 and RAD50 from the
thermophilic archaeon Pyrococcus furiosus. Our results
demonstrate that the P. furiosus Mre11 (pfMre11) and Rad50
(pfRad50) proteins show conservation within critical core domains and
form a large stable complex (pfMR50) that possesses both single-strand
endonuclease activity and double-strand exonuclease activity with a
3'-to-5' polarity. Despite the lack of overall sequence similarity, the
archaeal Mre11-Rad50 complex is functionally similar to those from
bacteria and eukaryotes.
| |
MATERIALS AND METHODS |
Identification and cloning of P. furiosus Mre11 and
Rad50.
The sequence of human Mre11 (hMre11; AAC78721) was used in
a BLAST search (tblastn at
http://www.ncbi.nlm.nih.gov/blast/unfinishedgenome.html) to identify a
homologue of hMre11 in the genome of P. furiosus. A single
hit with an E (expect) value of 3e-07 was found, and this reading frame
(pfMre11) was amplified by PCR from P. furiosus genomic DNA
using the oligonucleotide primers
5'-AAAAAAAAAAACATATGAAGTTTGCTCACTTAGCCGATATTC-3' and
5'-AAAAAAGGATCCCTATTATCTCGCACCACCAAGCCAGCTATC-3'. The
resulting PCR product was digested with BamHI and
NdeI and cloned into pET15b (Novagen) to create
pET15b-pfMre11.
To identify a probable homologue to human Rad50 (hRad50) in archaea, we
used the sequence of hRad50 (AAB07119) to perform a BLAST search (see
above) against available archaeal genomes. In general, we found two
hits per genome with E values in the range of 1e-26 and 1e-15 and which
spanned the entire sequence of 1,300 residues of hRad50. One hit was
generally annotated as "chromosome segregation protein," whereas
the other was annotated as "putative purine NTPase" (nucleoside
triphosphatase). In particular, we obtained two isolated hits with E
values of 6e-17 and 4e-17, respectively, in the genome of P. furiosus. Comparison with annotated homologues in other archaeal
genomes showed that the former hit has the highest homology with
chromosome segregation proteins whereas latter has the highest homology
with a putative purine NTPase. The gene for the latter (encoding
pfRad50) was found to be adjacent to the gene for pfMre11 in the
P. furiosus genome. This arrangement is similar to that
observed for the Mre11 and Rad50 homologues from E. coli,
SbcC and SbcD (14). Based on this, we hypothesized that this
putative purine NTPase from P. furiosus was the hRad50
homologue. The pfRad50 open reading frame was amplified by PCR from
P. furiosus genomic DNA using the oligonucleotide primers
5'-AAAAAAAAAAAACATATGAAATTGGAGAGAGTGACTGTGAAAAAC-3'
and 5'-AAAAAAAAACGGCCGCTATTAAGAGACCACCTCCACCTTGGAG-3'.
The resulting PCR product was digested with NdeI and
EagI and cloned into pET27b (Novagen) to create
pET27b-pfRad50.
Plasmid construction.
A segment of DNA carrying the ribosome
binding site (RBS), the start codon, and the coding sequence of pfMre11
was amplified by PCR using pET15b-pfMre11 as the template and
oligonucleotide primers
5'-AAAAAACGGCCGGATCCCGGGTACCGCGGCCGCGTCGAAATAATTTTGTTTAACTTTAAGAAGGAGAT-3' and 5'-GCTAGTTATTGCTCAGCGGTGGCAGCC-3'. The resulting
PCR product was digested with EagI and Bpu1102I
and ligated into pET27b-pfRad50 to create pET27b-pfRad50-pfMre11. This
bicistronic expression vector carries the T7 promoter, an RBS for
pfRad50, the open reading frame of pfRad50, a short linker, the RBS for
pfMre11, the open reading frame of pfMre11, and the T7 terminator. In
this construct, only pfMre11 contains a six-histidine tag, present on
the N terminus of the protein.
Expression and purification of the pfMR50.
Competent
E. coli BL12(DE3) cells were transformed with
pET27B-pfRad50-pfMre11; a single colony was used to inoculate 500 ml of
Luria broth containing kanamycin, (50 µg/ml) and this culture was
grown overnight. Then 120 ml of this overnight culture was used to seed
6 liters of Luria broth containing kanamycin sulfate (50 µg/ml), and
cells were grown at 37°C with shaking to an optical density at 550 nm
of 0.8. Protein expression was induced by the addition of
isopropyl-
-D-thiogalactopyranoside to a final
concentration of 0.8 mM, and the culture was further incubated at
37°C. After 5 h, the cells were harvested by centrifugation and
shock frozen in liquid nitrogen. To isolate the protein, the cells
(typically 20 g) were resuspended in 40 ml of a mixture containing
50 mM sodium phosphate (pH 8.0), 500 mM NaCl, 1 mM imidazole, and 1 tablet of Complete EDTA-free protease inhibitor mix (Roche Molecular Biochemicals). Cells were disrupted by sonication, and insoluble matter
was pelleted by centrifugation at 30,000 × g. The
supernatant was saved and heat shocked at 75°C for 10 min.
Precipitated protein was removed by centrifugation at 30,000 × g. The supernatant from this step was loaded on 5 ml of
Ni2+-nitrilotriacetic acid (NTA) resin (Qiagen, Carlsbad,
Calif.), and the column was washed with 50 ml of 50 mM phosphate (pH
8.0)-500 mM NaCl-1 mM imidazole followed by 50 ml of 20 mM phosphate
(pH 8.0)-200 mM NaCl-1 mM imidazole-5% glycerol. Bound protein was eluted with a linear gradient of 1 to 200 mM imidazole in 20 mM phosphate (pH 8.0)-200 mM NaCl-5% glycerol. Fractions from this column were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining; pfMre11 and
pfRad50 were found to coelute from this column. The pfMre11- and
pfRad50-containing fractions were pooled, titrated to pH 7.5, diluted
by 2 with water, and loaded onto 10 ml of phosphocellulose column
(Whatman) equilibrated with 20 mM Tris (pH 7.5)-100 mM NaCl-1 mM
EDTA-5% glycerol. The column was washed with 50 ml of equilibration
buffer, and the protein was eluted with a linear gradient from 10 to
250 mM phosphate in 20 mM Tris (pH 7.5)-100 mM NaCl-1 mM EDTA-5%
glycerol. pfMre11 and pfRad50 eluted at ~50 mM phosphate, and the
pfMre11- and pfRad50-containing fractions were pooled. The pool was
dialyzed against 20 mM Tris-500 mM NaCl-1 mM EDTA-5% glycerol. The
fractions from the phosphocellulose column contained significantly more
pfMre11 than pfRad50; thus, an aliquot of this pool was subjected to
further purification using a Superose 6 HR 10/30 column and a fast
protein liquid chromatography system. Proteins were injected on to a
Superose 6 column and separated in 50 mM Tris (pH 8.0)-300 mM NaCl-1
mM dithiothreitol (DTT)-10% glycerol at a flow rate of 18 ml/h.
Nuclease assays.
One microgram of purified pfMre11 or pfMR50
was incubated with 1 µg of circular single-stranded 
X174 DNA in 25 mM HEPES (pH 7.0)-25 mM NaCl-5 mM MnCl2 or 5 mM
MgCl2-1 mM DTT at 50°C; aliquots were removed at the
indicated times, and reactions were stopped by the addition of EDTA to
10 mM. One-tenth volume of 10× loading dye containing 3% SDS was
added to each aliquot, and the reaction products were separated on
1.0% agarose-1× Tris-borate-EDTA gels containing 500 ng of ethidium
bromide per ml.
Exonuclease activity was measured by monitoring the release of
trichloroacetic acid-soluble mononucleotides from restriction enzyme-linearized 3H-pUC19 DNA. Briefly,
3H-pUC19 was digested with HincII and used as
substrate in 300-µl reactions containing 25 mM HEPES-KOH (pH 7.0), 50 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 1 mM DTT, 1 nmol (total nucleotide) of 3H-pUC19, 1 mM ATP or
adenylyl-imidodiphosphate (AMP-PNP), and 100 nM pfMR50. Reactions were
performed at 50°C; 50-µl aliquots were removed, reactions were
stopped by the addition of EDTA to 10 mM, and the aliquots were placed
on ice. Following a 1-h incubation, 10 µl of salmon sperm DNA (2 mg/ml) and 100 µl of 10% trichloroacetic acid was added to each
aliquot. Nucleic acid was precipitated for 10 min on ice, the samples
were centrifuged for 10 min at 14,000 × g, and the
supernatants were assayed for the released 3H-nucleoside
monophosphate residues by liquid scintillation counting. Polarity was
determined by labeling a 39-mer oligonucleotide at the 5' end with T4
polynucleotide kinase (New England Biolabs, Beverly, Mass.) as
described by the manufacturer. The same oligonucleotide was 3'-end
labeled by incubation with terminal deoxynucleotidyltransferase (Roche
Biochemicals, Indianapolis, Ind.) in the presence of
[
-32P]ddATP (Amersham, Chicago, Ill.) as described by
the manufacturer. The labeled oligonucleotide was annealed to its
complement and used in exonuclease assays. Exonuclease assays contained
50 mM HEPES (pH 7.0), 50 mM NaCl, 10 mM MgCl2, 10 mM
MnCl2, 2 mM ATP, 1 mM DTT, 6 pmol of substrate
oligonucleotide, and 6 pmol of pfMR50. Reactions were incubated at
50°C, and aliquots were removed at various times, mixed with loading
dye containing 50% formamide, heat denatured, and resolved on 10%
polyacrylamide-7 M urea gels. Reaction products were visualized by autoradiography.
| |
RESULTS |
Isolation of the P. furiosus MRE11 and
RAD50 genes.
The P. furiosus homologue of
Mre11 was found by BLAST search, and the sequence coding for this
protein was similar to sequences of other Mre11 family members. pfMre11
is a 426-amino-acid protein with a predicted molecular mass of 49 kDa.
pfMre11 is 29% identical and 42% similar to hMre11 in the conserved
N-terminal domains of the two proteins. The four domains proposed to be
critical for nuclease activity are conserved in pfMre11, suggesting
that it possesses nuclease activity (Fig.
1A).
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FIG. 1.
Conservation of pfMre11 and pfRad50. (A) Alignment of
pfMre1 with homologues from humans (hMre11), S. cerevisiae
(scMre11), and E. coli (EcSbcD), using the program CLUSTALW.
The four conserved nuclease domains that have been described for the
Mre11 family (I, II, III, and IV) are shown with key residues in bold.
(B) Similar alignment of pfRad50, carried out using Rad50 homologues
from humans (hRad50), S. cerevisiae (scRad50), and E. coli (EcSbcC). The two conserved ATP binding domains are shown
with the key residues in bold (23a). Numbers indicate amino
acid positions of the motifs; numbers in parentheses are the number of
residues between conserved domains.
|
|
The gene encoding pfRad50 was found to be adjacent to the
pfMre11-coding sequence within the P. furiosus genome,
similar to that observed with the E. coli homologues SbcC
and SbcD (14). This Rad50 gene homologue codes for an
882-amino-acid protein with a predicted molecular mass of 104 kDa that
shows weak (19%) homology to the human protein. The key residues of
the Walker A and B ATP boxes are conserved between the putative pfRad50
and other Rad50 homologues from a variety of species (Fig. 1B)
(20). Analysis of pfRad50 and hRad50 with the program COILS
(www.ch.embnet.org/software/COILS_form.html) shows that the
central portion of each protein has a high probability to form an
alpha-helical coiled-coil structure (Fig.
2A). pfRad50 contains only two cysteine
residues that surprisingly occur in a conserved motif (Fig. 2B) at the
center of the coiled-coil domain. Despite overall low sequence homology
in this region, this motif is conserved in all Rad50 family members,
and the predicted probability of forming a coiled-coil structure at
these positions is zero. This suggests that this motif may be a hinge
region in the center of the coiled-coil domain.
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FIG. 2.
Structural conservation of pfRad50 and hRad50. (A) The
human and P. furiosus Rad50 homologues were examined for the
potential to form alpha-helical coiled-coil structures using the
program COILS (see Materials and Methods). The plot shows the
probability (P) of a given amino acid residue being present in an
alpha-helical coiled-coil domain and indicates that the overall
structure of the two proteins is conserved. The asterisk indicates the
position of a conserved CPXC motif in each protein. (B) Sequence
comparison of a number of Rad50 homologues shows the presence of a
conserved (Con) CPXC motif in the central portion of each molecule.
This region of the molecule is in a position that has a low probability
of forming a coiled-coil structure (A), implicating it as a potential
hinge region in the coiled-coil domain. Organisms listed are P. furiosus (Pf), Pyrococcus abyssi (Pa), E. coli (Ec), S. cerevisiae (Sc), Arabidopsis
thaliana (At), Caenorhabditis elegans (Ce), and
Homo sapiens (Hs).
|
|
Expression and purification of pfMR50.
To test for the
physical interaction of the two proteins, pfMre11 containing an
N-terminal histidine tag and pfRad50 were coexpressed in E. coli using a bicistronic vector that produces a single mRNA
encoding both proteins. After induction of expression, a soluble
extract was made and heated to 75°C to denature endogenous E. coli proteins. Following centrifugation to remove denatured proteins, this heat-soluble extract was loaded on to an
Ni2+-NTA-Sepharose column and bound proteins were eluted
with an imidazole gradient. Proteins in the eluted fractions were
separated by SDS-PAGE, and a band of the predicted molecular weight of
pfMre11 was observed by Coomassie blue gel staining along with a
coeluting band of the predicted molecular weight of pfRad50 (data not
shown). In this fraction, pfMre11 was present at a greater
concentration than pfRad50, suggesting that pfMre11 was expressed to a
greater level than pfRad50. To separate the pfMR50 complex from excess pfMre11, fractions from the Ni2+ column were pooled, and an
aliquot was subjected to gel filtration chromatography. Upon separation
on a Superose 6 column, the Ni2+ fraction gave rise to two
peaks, one containing the pfMR50 complex and the other containing only
pfMre11. This confirmed that pfMre11 is produced in excess in this
system (Fig. 3A). An aliquot of pfMR50
from the Superose 6 column was subjected to SDS-PAGE and Coomassie blue
staining. Quantitation of pfMre11 and pfRad50 indicates that the two
proteins are present in equimolar quantities (Fig. 3B). Thus, pfMre11
and pfRad50 form a heat-stable protein complex that appears to contain
multiple copies of each subunit in equimolar amounts.
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FIG. 3.
(A) Gel filtration analysis of pfMR50. pfMre11 and
pfRad50 were coexpressed in E. coli and purified by
Ni2+-affinity chromatography. This purified fraction was
separated on a Superose 6 gel filtration column. The first peak that
eluted from the gel filtration column is the coelution of the complex
of pfMre11 and pfRad50, while the second peak corresponds to the
elution of excess pfMre11. Comparison with native molecular weight
markers indicates a native molecular mass of 106 Da for
pfMR50. Purification through a heating step (see Materials and
Methods), Ni2+-NTA-Sepharose chromatography, and coelution
from a gel filtration column indicate that the two proteins exist in a
stable complex. (B) SDS-PAGE of pfMR50 and Coomassie blue staining
followed by quantitation of the bands by densitometry indicates that
pfMre11 and pfRad50 are present in equimolar amounts.
|
|
Characterization of pfMR50.
As other members of the Mre11
family have been reported to possess single-stranded DNA-specific
endonuclease activity, we examined pfMre11 for this type of activity
(4-6, 13, 15, 22). Purified protein were incubated at
50°C with single-stranded X174 DNA, and the amount of degradation
was assessed by gel electrophoresis and ethidium bromide staining.
Using pfMre11 alone, we found that the protein was able to efficiently
digest single-stranded DNA over the course of a standard 2-h incubation
in the presence of Mn2+ (Fig.
4). No digestion was observed in the
presence of Mg2+. We also examined the heterodimeric pfMR50
complex and found similar single-strand-specific endonuclease activity
(data not shown).
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FIG. 4.
Single-strand endonuclease activity of pfMre11. Based on
the presence of the conserved nuclease domains in the N terminus of
pfMre11, we examined the pfMre11 complex for nuclease activity.
Purified protein was incubated with X174 single-stranded DNA, and
the extent of single-strand endonuclease activity was assessed by gel
electrophoresis. The reactions were carried out in the presence of
either 5 mM MgCl2 (Mg2+) or 5 mM
MnCl2 (Mn2+). The results indicate that pfMre11
is a Mn2+-dependent single-strand endonuclease. Identical
results were obtained with pfMR50 (data not shown).
|
|
Studies of the E. coli Rad50 and Mre11 homologues SbcC and
SbcD, respectively, have shown that the digestion of linear DNA by this
complex requires the presence of ATP (4). Further work with
the human Mre11-Rad50-Nbs1 complex has shown that ATP is required for
the full spectrum of activities, including the ability to nick DNA
hairpins and the ability to unwind a short DNA duplex (16).
However, in both instances the hydrolysis of ATP could not be detected
by direct means. We also examined the digestion of linear plasmid DNA
by pfMR50. pfMR50 was able to digest linear plasmid DNA, and this
nuclease activity absolutely required the presence of a hydrolyzable
form of ATP (Fig. 5A). Reactions carried out in the presence of the nonhydrolyzable ATP analogue AMP-PNP failed
to show any appreciable release of mononucleotides (Fig. 5).
Furthermore, assays carried out in the absence of ATP did not show any
release of acid-soluble mononucleotides above background levels (data
not shown). Thus, the exonuclease activity of pfMR50 requires ATP
hydrolysis, and the small amount of digestion observed in the presence
of AMP-PNP but not in the absence of ATP suggests that ATP hydrolysis
is necessary for release following binding and digestion.
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FIG. 5.
Double-strand exonuclease activity of pfMR50. (A)
Purified pfMR50 was incubated with linear 3H-pUC19 plasmid
and 2 mM ATP (solid line) or 2 mM AMP-PNP (dashed line) for 1 h,
and aliquots were removed at the times indicated. The release of
acid-soluble mononucleotide at each time point was measured, and the
results indicate that the exonuclease digestion requires the presence
of ATP. Identical reactions done in the absence of ATP failed to show
any release of mononucleotide above background levels (data not shown).
(B) Purified pfMR50 was incubated with a double-strand oligonucleotide
substrate labeled at the 5' and 3' ends (as indicated), and reaction
aliquots were taken at the times indicated. Analysis of the products by
denaturing PAGE and autoradiography indicates the presence of a ladder
of reaction products with the 5'-labeled substrate, but no similar DNA
laddering is observed with a 3'-labeled substrate. Thus, pfMR50
functions as a 3'-to-5' exonuclease.
|
|
To examine the polarity of the exonuclease activity, an oligonucleotide
substrate was differentially labeled on the 5' and 3' ends and annealed
to its complement. The time course of digestion of this substrate
revealed the presence of exonuclease breakdown products with the
5'-labeled substrate but not with the 3'-labeled substrate, indicating
that the exonuclease proceeds in a 3'-to-5' manner (Fig. 5B).
| |
DISCUSSION |
The activities described in this paper for pfMR50 are similar to
those of the E. coli SbcCD complex and the human
Mre11-Rad50-Nbs1 complex (4, 5, 15, 22). Additionally, the
active site sequences of both pfRad50 and pfMre11 have been conserved
as well as the predicted coiled-coil domain of pfRad50 (20).
Thus, despite a lack of overall sequence conservation, it appears that
the structure and function of the Mre11-Rad50 complex have been
conserved throughout evolution. This is notable since there are very
few DNA repair proteins that are conserved across all three kingdoms
(1).
Biochemical analysis of the Mre11-Rad50 complexes from all three
kingdom has demonstrated that the complex possesses exonuclease activity with a 3'-to-5' polarity. In the case of the bacterial SbcCD
and pfMR50, the exonuclease activity requires the hydrolysis of ATP
(4). While this same experiment has not been described for
the human Mre11-Rad50-p95 complex, there are activities that are
stimulated by the addition of ATP, in particular, the ability of the
human complex to nick a fully complementary hairpin and to unwind a
short stretch of duplex DNA. We have observed a similar stimulation of
DNA binding by ATP for the catalytic domain of pfRad50 along with a
substantial conformational change (10). The binding of ATP
appears to generate a DNA binding channel in the structure of the
pfRad50 protein; given the inability of the human Mre11-Rad50-p95
complex to unwind DNA of longer than 34 bp, this suggests that DNA
binding by Rad50 in this channel leads to a local melting of small
stretches of duplex DNA. As the yeast Rad50 has been shown to have
ATP-dependent DNA binding activity, it appears that this mode of DNA
binding may be a universal characteristic of Rad50 from all species.
The molecular nature of the ATP-dependent DNA binding that leads to the
DNA melting is currently under investigation.
The conservation of the nuclease activity of Mre11 suggests a critical
conserved function. Although this nuclease activity was initially
thought to not be necessary for any mitotic function (13),
work with E. coli (5, 8a), and recent results for yeast implicate this activity in the metabolism of hairpin-containing DNA (19). Additionally, recent data have shown that a
mutation near the active site of hMre11 leads to ATLD, a disease
similar to ataxia telangiectasia (21). Although this
mutation, which changes asparagine 117 to serine, has not been examined
biochemically, its position adjacent to conserved active-site sequences
suggests that it affects nuclease activity. Furthermore, the addition
of a third component, Xrs2 in yeast and Nbs1 in humans, likely reflects the expansion of functionalities of the Mre11-Rad50 complex within eukaryotes. Unlike archaea and bacteria, eukaryotic systems have the
additional burden of sexual reproduction, cell cycle control, and
persistent DNA ends in the form of telomeres. Nbs1 and Xrs2 may have
evolved to carry out these eukaryote-specific functions.
The details of the in vivo functions of the Mre11-Rad50 complex are not
clearly understood. pfMR50 has been observed to be a dumbbell-like
molecule similar to other SMC family members (10, 11). This
suggests that the Mre11-Rad50 complex plays a structural role in its
DNA repair and recombination functions. A general premise of DNA DSB
repair is that the two free DNA ends must be maintained in reasonable
proximity to one another for accurate repair to take place. This
premise is probably true regardless of whether a particular break is
being repaired by homologous recombination or nonhomologous end
joining. Additionally, in homologous recombination the template for
repair is typically the sister chromatid, and recent genetic results
have demonstrated a function for Mre11 and Rad50 in mediating sister
chromatid interactions (2, 25). Therefore, the dumbbell-like
molecules of Mre11-Rad50 could be crucial for the interaction of sister
chromatids or DNA ends in order to allow proper repair to take place.
We thank the members of the Tainer and Carney laboratories for
helpful discussions. We also thank R. DiGate and A. Cseresnyes for the
generous gift of the 3H-pUC19 DNA.
This work was supported by Laboratory Directed Research and Development
funds of LBNL (J.A.T. and J.P.C.) and American Cancer Society research
project grant RPG-00-059-01-CCE (to J.P.C.). K.P. was supported by a
BASF fellowship through the Studienstiftung des Deutschen Volkes, and
J.P.C. was supported in part by NIH grant GM53918 (to William F. Morgan).
| 1.
|
Aravind, L.,
D. R. Walker, and E. V. Koonin.
1999.
Conserved domains in DNA repair proteins and evolution of repair systems.
Nucleic Acids Res.
27:1223-1242[Abstract/Free Full Text].
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| 2.
|
Bressan, D. A.,
B. K. Baxter, and J. H. Petrini.
1999.
The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae.
Mol. Cell. Biol.
19:7681-7687[Abstract/Free Full Text].
|
| 3.
|
Carney, J. P.,
R. S. Maser,
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Abstract
Introduction
