To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.
The Werner syndrome and the Nijmegen breakage syndrome are recessive genetic disorders that show increased genomic instability, cancer predisposition, hypersensitivity to mitomycin C and ␥-irradiation, shortened telomeres, and cell cycle defects. The protein mutated in the premature aging disease known as the Werner syndrome is designated WRN and is a member of the RecQ helicase family. The Nbs1 protein is mutated in Nijmegen breakage syndrome individuals and is part of the mammalian Mre11 complex together with the Mre11 and Rad50 proteins. Here, we show that WRN associates with the Mre11 complex via binding to Nbs1 in vitro and in vivo. In response to ␥-irradiation or mitomycin C, WRN leaves the nucleoli and co-localizes with the Mre11 complex in the nucleoplasm. We detect an increased association between WRN and the Mre11 complex after cellular exposure to ␥-irradiation. Small interfering RNA and complementation experiments demonstrated convergence of WRN and Nbs1 in response to ␥-irradiation or mitomycin C. Nbs1 is required for the Mre11 complex promotion of WRN helicase activity. Taken together, these results demonstrate a functional link between the two genetic diseases with partially overlapping phenotypes in a pathway that responds to DNA double strand breaks and interstrand cross-links.DNA damage in the form of double strand breaks (DSBs) 1 or cross-links compromises the integrity of cells. Defects in repairing these lesions are associated with human chromosome fragility syndromes such as Werner syndrome (WS), Nijmegen breakage syndrome (NBS), ataxia telangiectasia (AT), and ataxia telangiectasia-like disorder (ATLD). In contrast to WS, NBS and ataxia telangiectasia-like disorder (with Mre11 mutations) are defective in checkpoint responses and show clinical features of immunodeficiency, hyperpigmentation spots, and ovarian dysgenesis. Noticeably, in addition to the aforementioned cellular similarities, both WS and NBS display graying of the hair, short stature, a "bird-like" face, increased cancer susceptibility, and reduced life span (1-6). Loss of fidelity in repairing DNA DSBs results in chromosomal rearrangements (7), a defect that is prominent in both WS and NBS. The proteins mutated in these syndromes are implicated in the repair of DNA DSBs and interstrand cross-links (ICLs) (2,4,5,8,9). Vertebrate Nbs1 is essential for repairing DNA DSBs by homologous recombination (10), and WRN participates in this repair pathway by resolving the recombinational intermediates (11). WS cells are more sensitive to DNA cross-linkers than to any other genotoxic drugs and are defective in repairing DNA ICLs in vivo (2, 8). WRN exhibits 3Ј 3 5Ј helicase, 3Ј 3 5Ј exonuclease, and ATPase activities (12, 13). Although Nbs1 has no known enzymatic activity, it is required for optimal exonuclease and endonuclease activities of the Mre11 complex and for the correct nuclear localization of Mre11 and Rad50 proteins (4, 14). The Mre11 complex exhibits clear 3Ј 3 5Ј exonuclease activity but demonstrates only limited DNA unwindin...
The Mre11, Rad50 and Nbs1 proteins make up the conserved multi-functional Mre11 (MRN) complex involved in multiple, critical DNA metabolic processes including double-strand break repair and telomere maintenance. The Mre11 protein is a nuclease with broad substrate recognition, but MRN-dependent processes requiring the nuclease activity are not clearly defined. Here, we report the functional and structural characterization of a nuclease-deficient Mre11 protein termed mre11-3. Importantly, the hmre11-3 protein has wild-type ability to bind DNA, Rad50 and Nbs1; however, nuclease activity was completely abrogated. When expressed in cell lines from patients with ataxia telangiectasia-like disorder (ATLD), hmre11-3 restored the formation of ionizing radiation-induced foci. Consistent with the biochemical results, the 2.3 A crystal structure of mre11-3 from Pyrococcus furiosus revealed an active site structure with a wild-type-like metal-binding environment. The structural analysis of the H85L mutation provides a detailed molecular basis for the ability of mre11-3 to bind but not hydrolyze DNA. Together, these results establish that the mre11-3 protein provides an excellent system for dissecting nuclease-dependent and independent functions of the Mre11 complex.
T he process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G 2 /M "arrest," showed that p21Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53 -/-mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21 -/-mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.
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