Mutagenesis Advance Access originally published online on April 9, 2007
Mutagenesis 2007 22(4):269-274; doi:10.1093/mutage/gem011
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Dimerization of the Rad50 protein is independent of the conserved hook domain
The Radiation Oncology Research Laboratory and The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, BRB 6-011, 655 West Baltimore Street, Baltimore, MD 21201, USA
The Mre11 complex (Mre11–Rad50–Nbs1) is involved in a diverse array of DNA metabolic processes including the response to DNA double-strand breaks (DSBs). The structure of Rad50 plays a key role in the DNA-binding and end-bridging activity of the complex. An interesting feature within the central portion of the Rad50 protein is the Rad50 hook region that is defined by the highly conserved CXXC motif. The structure of the Pyrococcus furiosus Rad50 hook region revealed an intermolecular dimerization of Rad50 through the coordination of a zinc ion by the four cysteines. Biochemical and genetic analysis in Saccharomyces cerevisiae have shown that mutations in the conserved cysteines impact all functions of the Mre11 complex including interaction with Mre11, increased sensitivity to DSB inducing agents, telomere maintenance and intrachromosomal association. Mutations in the yeast hook domain can lead to increased chromosome fragmentation, suggesting that the hook domain of Rad50 is essential for the tethering of chromosome ends. In this study, we have examined the effects of mutating the key cysteine residues in the hook domain of human Rad50 (hRad50), focusing on the interactions Rad50 has with itself, Mre11 and DNA. Our results reveal that mutation of the conserved cysteine residues abrogates dimerization at the hook domain in hRad50; however, disrupting dimerization at this domain does not appear to impair the interaction of full-length hRad50 with itself and hMre11 or affect DNA-binding activity of the hMre11–Rad50 complex.
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Introduction |
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A double-strand break (DSB) in DNA can result in the disruption of genetic information through gene deletions, translocations and missegregation of chromosomes if not repaired properly (1
–3). These mutations can ultimately lead to genomic instability. The Mre11 complex plays a key role in maintaining genome stability. It is involved in many diverse DNA metabolic processes, which include the damage response and repair of DSBs, as well as telomere maintenance [reviewed in refs. (4–6)]. The human Mre11 (hMre11) complex is composed of the Mre11, Rad50 and Nbs1 proteins. Mre11 and Rad50 are conserved from archea to mammals, while Nbs1-like homologs have not been found in prokaryotes or archea, suggesting that the third component of the complex may only be found in eukaryotes (7). Biochemical analyses of the individual proteins have revealed nuclease activity in Mre11 and ATPase activity in Rad50. The Nbs1 subunit appears to function as a protein–protein interaction module as no enzymatic function has yet been ascribed to it [reviewed in refs. (4–6)]. Using atomic force microscopy (AFM), the complex has been shown to display DNA end-bridging activity that is independent of nuclease activity and ATP, suggesting a structural role in DNA repair (8–11). The yeast complex, Rad50–Mre11–Xrs2 plays an essential role in holding DNA ends together, thus preventing the missegregation of acentric chromosome fragments during cell division (12,13). Notably, a mutation disrupting the yeast Rad50 hook domain prevented intrachromosomal association, generating an increase in chromosome fragmentation (13).
Rad50 shares similar function and structure to proteins belonging to the structural maintenance of chromosome (SMC) family (14,15). As the name suggests, SMCs play a critical role in the organization of chromosomes, including chromosome segregation and recombinational repair (16,17). SMCs also share a common unique protein architecture consisting of a long central coiled-coil segment flanked by nucleotide-binding motifs known as Walker A and Walker B. The coiled-coil segments are antiparallel, and upon folding back on one another, the Walker A and Walker B motifs are brought together to create an ATP-binding domain which is considered the ‘head’ of the protein. The apical end where the coiled-coils fold is referred to as the ‘hinge’ domain in SMCs (18). Rad50 also consists of the coiled coils and Walker A and B head domain; however, rather than having a hinge region at the apex of the coiled coils, it contains a highly conserved CXXC motif that is responsible for zinc-dependent dimerization (19,20). The crystal structure of the conserved central region of the Pyrococcus furiosus Rad50 revealed an intermolecular arrangement of Rad50 through coordination of a zinc ion by the four conserved cysteines, two from each CXXC motif (19,21). The composite zinc finger has a unique ‘hook’ shape leading us to refer to this as Rad50 hook. Mutating the cysteines to glycines in the Saccharomyces cerevisiae hook domain disrupted complex formation with Mre11, indicating defects in repair as observed by the increase in ionizing radiation sensitivity (20). Interestingly, Mre11 interacts with the Rad50 coiled coil at the opposite end from the hook region at the base of the head domain (22). In addition to Mre11 binding, the head domain of Rad50 is thought to mediate much of the DNA-binding and end-bridging activity (8–11). Thus, the hook domain may be involved in regulating DNA binding or end bridging through the coiled coils.
In order to understand the biological contributions of the hook domain to complex formation and the function of the hMre11 complex, we have generated full-length human Rad50 (hRad50-fl) protein as well as the hRad50 hook (hRad50-hk) domain that contain mutations in the CXXC motif. We investigated the effects of mutating the key cysteine residues within the hRad50-hk domain on complex formation with hMre11 and interaction with itself. Our results reveal that, while the conserved cysteine residues are required for dimerization at the hook domain, these mutations do not appear to impair the interaction of hRad50 with itself, hMre11 or DNA binding. To distinguish between full-length and hook domain Rad50 protein and the various mutations discussed throughout this text, we have used abbreviations that are listed and described in Figure 1.
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Plasmid expression constructs and mutagenesis
The human hook protein construct JL443 (pET-29c-hook) was a gift from J.A.Tainer (Scripps Research Institute, La Jolla, CA). The Rad50 sequence from JL443 (Rad50 cDNA 1882–2361nt) was cloned into the pET-duet vector (Novagen, San Diego, CA) using NdeI and XhoI restriction sites excluding a histidine tag (pET-HK). The Rad50 cDNA 1906–2208nt was cloned into the pACYC-duet vector (Novagen) using BamHI and HindIII restriction sites including an N-terminal histidine tag (pACYC-HK-his). The hMre11-fl and Rad50 cDNAs were gifts from J.H.J. Petrini (Memorial Sloan-Kettering Cancer Center, New York, NY). These genes were cloned into pFastBac-1 (Invitrogen, Carlsbad, CA). The hMre11 construct included a 6xHis tag at the C-terminus. To generate full-length Rad50 constructs containing a C-terminal HA or FLAG tag, primers were designed to amplify the C-terminus of Rad50 from 2385–3936nt and include an HA or FLAG tag before the stop codon. The polymerase chain reaction (PCR) product was cloned into pFB1-Rad50 using BlpI and XbaI restriction sites to create hRad50-fl with the C-terminal tags. QuikChange site-directed mutagenesis XL (Stratagene, La Jolla, CA) was used to generate specific mutations in Rad50. Single and double mutations were generated using primers designed to include either the single- or double-point mutation. The triple mutation was generated by using the double mutant construct as a template and primers containing all three point mutations. Primer sequences are available upon request. All sequences were verified at the Biopolymer Laboratory at the University of Maryland, School of Medicine in Baltimore, MD, USA.
Protein purification for antiserum
Purified hook protein was used to generate antiserum specific to 628–787 amino acids of hRad50 (Strategic Biosolutions, Newark, DE), which is now available through Novus Biologicals, Littleton, CO. BL21-CodonPlus (DE3) RIL cells (Stratagene) containing the pET-HK construct were induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) and expressed in the presence of 50 µM ZnSO4 for 4 h at 37°C. Cells were lysed by sonication in lysis buffer [100 mM Tris, pH 8.0, 150 mM NaCl, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail] and spun down at 14 000x g for 20 min at 4°C. The clarified extract was passed through a coupled SP/Q column (Amersham, Piscataway, NJ), followed by the removal of the SP column. Protein bound to the Q column was eluted using a salt gradient from 100 mM to 1 M NaCl. Fractions were further purified using HiPrep Sephacryl S300 (Amersham) in the presence of TDG (20 mM Tris, pH 7.4, 1 mM DTT, 5% glycerol and 150 mM NaCl). Fractions containing purified protein were stored in small aliquots at –80°C. Protein concentrations were determined by Bradford assay. To test for Rad50 detection in cellular extract, HeLa cells were lysed in 500 mM NaCl, 50 mM Tris, pH 8.0, 1% igepal, 20 mM ß-mercaptoethanol, 10% glycerol, 1 mM NaOV and 10 mM NaF, iced for 30 min, passed through syringe three times to lyse and spun down for 20 min at 14 000x g at 4°C.
Nickel pull-down assays
pET-HK and pACYC-HK-his constructs were co-transformed in BL21-DE3 (Novagen) and expressed in the same conditions as listed for pET-HK. The cells were lysed by sonication in buffer A (20 mM NaPO4, pH 7.4, 500 mM NaCl and 20 mM ß-mercaptoethanol) plus protease inhibitor cocktail and 20 mM imidazole. Cellular extract (250 µg) was bound to 100 µl Ni-NTA beads (Qiagen, Valencia, CA) for 10 min at 4°C. The beads were washed three times with 0.5 ml buffer A + 50 mM imidazole and eluted with 100 µl buffer A + 500 mM imidazole. Soluble crude extract (input) and imidazole elute were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Coomassie staining and western blot analysis using antibodies specific to Rad50 hook and Mre11 (Novus Biologicals). Bound protein was quantified using AlphaEase Chemi Imager (Alpha Innotech Corp., San Leandro, CA). The guanidine–HCl washes were performed the same way, but included an additional wash step using 0.5 ml buffer A + 50 mM imidazole + 4 M GuHCl, followed by elution using 500 mM imidazole.
Full-length Rad50 and Mre11 were expressed in Sf9 insect cells for 48 h using recombinant baculovirus generated from bacmids according to Bac-to-Bac instructions (Invitrogen). Cell pellets were re-suspended in buffer A that included protease inhibitor cocktail and 20 mM imidazole, passed through 25 G5/8 needle and spun down at 37 000 r.p.m. for 1 h at 4°C using a 70Ti rotor in the Beckman Coulter, LE-80K ultracentrifuge. The clarified extract was bound to Ni-NTA agarose beads (Qiagen) for 30 min at 4°C followed by the wash and elution steps described for the hook interaction studies. Full-length hRad50–Mre11 complexes were further purified using HiPrep Sephacryl S300 as described for the hook protein used for antiserum.
Co-immunoprecipitation
Sf9 insect cells were co-infected with Rad50-HA, Rad50-FLAG and Mre11-his and expressed for 48 h. Cell pellets re-suspended in Sf9 IP buffer (50 mM Tris, pH 7.4, 5% glycerol, 150 mM NaCl, 1% igepal and protease inhibitor cocktail) lysed and clarified as described for pull-down assay. Soluble crude extract (500 µg) was incubated with HA, FLAG or no antibody (Sigma, St. Louis, MO) for 1 h at 4°C, followed by the addition of protein A/G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for an additional hour and washed with Sf9 IP buffer. Protein was released from the beads by boiling in 0.1% SDS, separated by SDS-PAGE and transferred to nitrocellulose for 1 h at 100 V. The membranes were blocked in 5% milk/TBST (100 mM Tris, pH 7.5, 300 mM NaCl, 0.1% Tween-20) for 1 h. Primary (anti-FLAG 1:500, anti-Mre11 1:1000) and secondary antibodies were each incubated for 1 h at room temperature and detected using LumiGLO chemiluminescence (KPL).
DNA-binding assays
Biotinylated double-strand DNA 1-kbp substrate (10 nM) was bound to streptavidin-coated magnetic beads as per the manufacturer's instructions (Dynabeads M-280, Dynal Biotech, Invitrogen, Carlsbad, CA). The DNA–bead complex was equilibrated with buffer TDA-b (50 mM Tris, pH 7.4, 5% glycerol, 0.5% igepal, 100 mM NaCl and 1 mM DTT) followed by the addition of 60 nM protein for a total volume of 50 µl and incubated for 30 min on ice. A magnet was used to separate the bead–DNA–protein complex from any unbound protein found in the supernatant. The bead–DNA–protein complex was washed twice in 0.5 ml buffer TDA-w (50 mM Tris, pH 7.4, 5% glycerol, 0.5% igepal and 2.5 mM MgCl2) and released from the beads by boiling in 0.1% SDS. Western blot analysis was used to detect the specified protein.
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Generation and characterization of antiserum specific to the hRad50-hk domain
To investigate the hRad50-hk domain, we expressed and purified a fragment of the hRad50 protein that was composed of
150 amino acids located within the central region of Rad50 including the CXXC zinc-binding motif and some of the coiled-coil domain (Figure 1: hRad50-hk; Figure 2A). The soluble crude extract was purified using coupled SP/Q ion exchange chromatography followed by gel filtration (Figure 2B). The purified protein was used to generate antiserum specific to the hRad50-hk domain. In addition to recognizing the hRad50-hk protein, western blot analysis using purified full-length hMre11–hRad50 complex and whole-cell extract from HeLa cells revealed that the hook antiserum recognizes hRad50-fl, as well (Figure 2C).
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Mutations in the hook domain significantly disrupt dimer formation
To characterize the interaction of hRad50 at the hook domains, we wanted to confirm that the zinc-dependent dimerization observed in P. furiosus was dependent on the same CXXC motif found in humans. To accomplish this, we co-expressed affinity-tagged and non-tagged hRad50-hk proteins of different sizes so that they could be separated and analyzed by SDS-PAGE. The non-tagged proteins were composed of
160 amino acids and ran at a molecular weight of 18.5 kDa. The affinity-tagged hRad50-hk protein was a smaller region composed of 100 amino acids, including an N-terminal histidine tag and ran at a molecular weight of 13.5 kDa (Figure 2A). The tagged and non-tagged vectors were co-transformed and expressed together in Escherichia coli for 4 h at 37°C. To make sure the zinc-dependent dimerization was not affected by protein over-expression, we expressed in the presence of 50 µM ZnSO4. Nickel pull-down assays were performed to capture the tagged hook protein. By comparing the amount of the non-tagged protein that came down with the tagged protein, we were able to determine the interaction between the two proteins. Our results for the wild-type hRad50-hk proteins displayed equal amounts of the tagged and non-tagged protein indicating hRad50-hk dimer formation (Figure 3A and B).
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Using site-directed mutagenesis, we constructed mutations in the zinc-binding motif, CXXC, changing the first, last or both cysteines to a glycine [Figure 1: C1G, C2G, C(1,2)G]. This was carried out with both the tagged and non-tagged hook proteins. The mutated tagged and mutated non-tagged hook proteins were co-transformed and expressed together followed by nickel pull-down assays to capture the tagged mutant protein and separated by SDS-PAGE. In contrast to the wild-type hRad50-hk data, the amount of the non-tagged mutant protein found in the pull-down assay was significantly lower when compared to its tagged counterpart (Figure 3A and B). Our results revealed that the mutations in any of the conserved cysteines in the hook domain disrupted dimer formation. In addition, we did not observe a significant difference between a single- or double-cysteine mutation, suggesting that each cysteine in the CXXC motif is individually required for dimerization (Figure 3A and B). Under these conditions, we were able to disrupt
80% of the interaction between the tagged and non-tagged hRad50-hk proteins. Unlike P. furiosus, the Rad50 proteins found in humans and yeasts contain an additional cysteine immediately N-terminal to the CXXC motif (20). We generated a triple-cysteine mutation [Figure 1: C(0,1,2)G] to verify that the non-conserved cysteine was not partially rescuing the 20% of the hook interaction that could not be disrupted. The pull-down assay results revealed the same amount of dimer disruption as observed for the single- and double-cysteine mutations (Figure 3A and B). To confirm that the dimer disruption was a result of altering the cysteine zinc-binding site, we mutated a conserved arginine residue adjacent to the CXXC motif that is not required for zinc-dependent dimerization (Figure 1: R686A). The interaction between the R686A mutant proteins is similar to the wild-type hRad50-hk, indicating that dimerization is mediated through the conserved CXXC motif of the hRad50-hk domain (Figure 3A and B). As a control, the hRad50-hk proteins were expressed and purified individually to check for non-specific binding (Figure 3C). We did not detect any of the non-tagged proteins in the imidazole elutions (data not shown for the double and triple mutations). To be certain that our results were not affected by the chelation of zinc from using a 6xHis tag, we performed pull-down assays using GST-tagged Rad50-hk co-expressed with the non-tagged hRad50-hk. Similar to Figure 3, single and double mutations in the conserved cysteine residues significantly inhibited dimerization (data not shown).
hRad50-fl hook mutants reveal interaction with hMre11
Mutational analysis of CXXC domain of S. cerevisiae reveal that a C2G mutation in full-length Rad50 disrupts interaction with Mre11, suggesting a defect in the Mre11-binding site found at the head domain of Rad50. To determine if complex formation with hRad50 was affected, we co-expressed the hRad50-fl with hMre11 and checked for interaction. Sf9 insect cells were infected with recombinant baculovirus particles expressing 6xHis-tagged hMre11 with hRad50-fl WT, C1G, C2G and C(1,2)G. hMre11 was separated from the cellular extract by nickel pull-down assays and checked for the presence of hRad50-fl using western blot analysis. The amount of Rad50 found in the imidazole elution was compared to the amount of eluted Mre11 (Figure 4A, top panel, and 4B). The results revealed that all hRad50-fl hook domain mutants could still interact with Mre11. Furthermore, the hMre11–hRad50 complexes were washed using buffer containing 4 M GuHCl and we still observed interaction between hMre11 and hRad50-fl hook domain mutants (Figure 4A, bottom panel, and 4B). In contrast to the yeast Rad50 protein (20), hRad50 mutated in the conserved CXXC motif is able to strongly interact with hMre11.
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hRad50-fl hook mutants reveal interaction with each other in the absence of hMre11
We next examined if hRad50-fl could interact with itself at the head domain when dimerization at the hook domain was disrupted. To detect interaction between two hRad50-fl molecules, we generated recombinant baculovirus expressing hRad50-fl with an HA tag or a FLAG tag which were co-expressed in the absence or presence of hMre11. The cellular extracts were immunoprecipitated with either HA or Flag antibody and probed with FLAG antibody. Interaction between the wild-type HA-tagged hRad50-fl and FLAG-tagged hRad50-fl were observed in the absence and presence of hMre11 (Figure 4C). Though the detection of FLAG-tagged hRad50 in the input was extremely faint, our immunoprecipitation indicates expression and interaction of the two hRad50 proteins. This was repeated using the hRad50-fl hook mutated in both the conserved residues and the results were similar to the wild-type hRad50-fl. The HA-tagged hRad50-fl-C(1,2)G and FLAG-tagged hRad50-fl-C(1,2)G could interact in the absence and presence of Mre11 (Figure 4D). We could not detect a difference in binding in the absence or presence of hMre11, indicating that the observed hRad50 interaction is independent of Mre11 and the hook domain.
Full-length Mre11/Rad50 hook mutants interact with DNA
To determine if dimerization at the Rad50 hook domain has any effect on DNA-binding activity, we performed DNA pull-down assays using purified hMre11–Rad50-fl [WT, C1G or C(1,2)G] complexes. The DNA substrate was a 1-kbp PCR product biotinylated at one end that could bind to streptavidin-coated magnetic beads. The DNA–bead complex was incubated with the indicated hMre11–Rad50-fl protein complex followed by the capture of the protein–DNA–bead complex using a magnet and washing away any unbound protein. Bound protein was eluted from the DNA–bead complex and detected by western blot using antiserum specific to each protein of the complex. The results from the pull-down assays showed that almost half of the input protein was bound to DNA (Figure 5). The percentage of Rad50 bound to DNA when compared to the original input concentration was calculated for each hMre11–Rad50-fl complex and plotted together for comparison (Figure 5B). Under these assay conditions, we were not able to detect a significant difference in DNA-binding activity. These results show that dimerization of the Rad50 hook domain is not necessary for DNA binding.
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Discussion |
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hRad50 does not require the CXXC motif to interact with itself or Mre11
To examine the interaction between the hRad50 CXXC motifs, we first over-expressed hRad50-hk in E. coli. Mutation of one or all the cysteines involved in binding zinc caused significant dimer disruption at the hook domain; however, we consistently observed
20% of the non-tagged protein interacting with the tagged protein. We believe that this is a result of the over-expression of the hook proteins in E. coli. A combination of the high level of protein expression that occurs in E. coli and the intrinsic nature of the protein to coil upon itself may be generating an intermolecular interaction at the coiled coils that is independent of the zinc-mediated CXXC motif. Alternatively, there may be some minor non-conserved interaction mechanism responsible for the residual binding we observed.
At the same time as our studies, the functional significance of the hook domain was analyzed in S. cerevisiae using a system in which the entire hook domain was removed and replaced with a ligand-inducible FKBP dimerization cassette (21). In agreement with the human data, the removal of the hook domain did not have an effect on Mre11 interaction. Yeast strains that were ‘hook minus’ displayed phenotypes similar to rad50-null mutants, such that they showed defects in DNA repair and telomere maintenance, even though the complex was observed to bind chromatin similar to rad50-wild-type strain (21). Using their conditional dimerization module, Wiltzius et al. (21) could restore interaction at the hook domain, resulting in the partial suppression of the mutant phenotypes. Their results reveal that the hook region is critical for DNA repair, telomere maintenance and DSB formation during meiotic recombination.
Our analysis of hRad50-fl reveals that the cysteines found in the hook domain are not required for hRad50 interaction with itself or hMre11 and has no obvious effect on DNA-binding activity. Dimerization of hRad50-fl appears to involve more than just a functional hook domain. This data are consistent with the physical observations of the hMre11 complex using AFM imaging. The coiled coils of hRad50 appear to be flexible and dynamic. Using AFM to determine the distribution of different conformations of human R2M2 heterotetramers revealed that 60% of the complexes formed an open V-shape that were connected via the head domain (11). The remaining complexes formed closed circular structures that seemed to interact at the head and hook domains. Interestingly, the hMre11–Rad50 complexes were never observed to form an open V-shape connected at the hook domain (11). The distribution of open to closed complexes and time-resolved high-resolution SFM imaging suggest that the coiled coils are flexible and do not require hook interaction at all times, suggesting that this dimerization event could be highly regulated in response to DNA damage (8,10,11). In agreement with this, we observed mutations that disrupted the zinc-dependent binding at the hook domain were still able to interact with each other independent of the presence of hMre11. These results suggest that hRad50 may interact with itself through multiple contacts.
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Acknowledgments |
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The authors wish to thank the members of the Carney laboratory for helpful discussions and comments on the manuscript. This work was supported by grant CA87851 (J.P.C.) and CA92584 (J.P.C. and J. A. Tainer) from the National Cancer Institute.
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* To whom correspondence should be addressed. Tel: +1 410 706 4276; Fax: +1 410 706 6138; Email: jcarney{at}som.umaryland.edu
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1. van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat. Rev. Genet. (2001) 2:196–206.[CrossRef][Web of Science][Medline]
2. Rich T, Allen RL, Wyllie AH. Defying death after DNA damage. Nature (2000) 407:777–783.[CrossRef][Medline]
3. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature (2001) 411:366–374.[CrossRef][Medline]
4. Assenmacher N, Hopfner KP. MRE11/RAD50/NBS1: complex activities. Chromosoma (2004) 113:157–166.[Web of Science][Medline]
5. D'Amours D, Jackson SP. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling. Nat. Rev. Mol. Cell. Biol. (2002) 3:317–327.[CrossRef][Web of Science][Medline]
6. van den Bosch M, Bree RT, Lowndes NF. The MRN complex: coordinating and mediating the response to broken chromosomes. EMBO Rep. (2003) 4:844–849.[CrossRef][Web of Science][Medline]
7. Connelly JC, Leach DR. Tethering on the brink: the evolutionarily conserved Mre11-Rad50 complex. Trends Biochem. Sci. (2002) 27:410–418.[CrossRef][Web of Science][Medline]
8. de Jager M, van Noort J, van Gent DC, Dekker C, Kanaar R, Wyman C. Human Rad50/Mre11 is a flexible complex that can tether DNA ends. Mol. Cell (2001) 8:1129–1135.[CrossRef][Web of Science][Medline]
9. Trujillo KM, Roh DH, Chen L, Van Komen S, Tomkinson A, Sung P. Yeast xrs2 binds DNA and helps target rad50 and mre11 to DNA ends. J. Biol. Chem. (2003) 278:48957–48964.
10. Moreno-Herrero F, de Jager M, Dekker NH, Kanaar R, Wyman C, Dekker C. Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA. Nature (2005) 437:440–443.[CrossRef][Medline]
11. de Jager M, Trujillo KM, Sung P, Hopfner KP, Carney JP, Tainer JA, Connelly JC, Leach DR, Kanaar R, Wyman C. Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex. J. Mol. Biol. (2004) 339:937–949.[CrossRef][Web of Science][Medline]
12. Kaye JA, Melo JA, Cheung SK, Vaze MB, Haber JE, Toczyski DP. DNA breaks promote genomic instability by impeding proper chromosome segregation. Curr. Biol. (2004) 14:2096–2106.[CrossRef][Web of Science][Medline]
13. Lobachev K, Vitriol E, Stemple J, Resnick MA, Bloom K. Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex. Curr. Biol. (2004) 14:2107–2112.[CrossRef][Web of Science][Medline]
14. Haering CH, Lowe J, Hochwagen A, Nasmyth K. Molecular architecture of SMC proteins and the yeast cohesin complex. Mol. Cell (2002) 9:773–788.[CrossRef][Web of Science][Medline]
15. Hopfner KP, Tainer JA. Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures. Curr. Opin. Struct. Biol. (2003) 13:249–255.[CrossRef][Web of Science][Medline]
16. Hirano T. SMC protein complexes and higher-order chromosome dynamics. Curr. Opin. Cell. Biol. (1998) 10:317–322.[CrossRef][Web of Science][Medline]
17. Hirano M, Hirano T. ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer. EMBO J. (1998) 17:7139–7148.[CrossRef][Web of Science][Medline]
18. Hirano T. At the heart of the chromosome: SMC proteins in action. Nat. Rev. Mol. Cell. Biol. (2006) 7:311–322.[CrossRef][Web of Science][Medline]
19. Hopfner KP, Karcher A, Shin DS, Craig L, Arthur LM, Carney JP, Tainer JA. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell (2000) 101:789–800.[CrossRef][Web of Science][Medline]
20. Hopfner KP, Craig L, Moncalian G, et al. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature (2002) 418:562–566.[CrossRef][Medline]
21. Wiltzius JJ, Hohl M, Fleming JC, Petrini JH. The Rad50 hook domain is a critical determinant of Mre11 complex functions. Nat. Struct. Mol. Biol. (2005) 12:403–407.[CrossRef][Web of Science][Medline]
22. Hopfner KP, Karcher A, Craig L, Woo TT, Carney JP, Tainer JA. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell (2001) 105:473–485.[CrossRef][Web of Science][Medline]
Received on January 9, 2007; revised on February 8, 2007; accepted on February 9, 2007.
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