The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, 'bird-like' face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).

Ataxia-oculomotor apraxia (AOA1) is a neurological disorder with symptoms that overlap those of ataxia-telangiectasia, a syndrome characterized by abnormal responses to double-strand DNA breaks and genome instability. The gene mutated in AOA1, APTX, is predicted to code for a protein called aprataxin that contains domains of homology with proteins involved in DNA damage signalling and repair. We demonstrate that aprataxin is a nuclear protein, present in both the nucleoplasm and the nucleolus. Mutations in the APTX gene destabilize the aprataxin protein, and fusion constructs of enhanced green fluorescent protein and aprataxin, representing deletions of putative functional domains, generate highly unstable products. Cells from AOA1 patients are characterized by enhanced sensitivity to agents that cause single-strand breaks in DNA but there is no evidence for a gross defect in single-strand break repair. Sensitivity to hydrogen peroxide and the resulting genome instability are corrected by transfection with full-length aprataxin cDNA. We also demonstrate that aprataxin interacts with the repair proteins XRCC1, PARP-1 and p53 and that it co-localizes with XRCC1 along charged particle tracks on chromatin. These results demonstrate that aprataxin influences the cellular response to genotoxic stress very likely by its capacity to interact with a number of proteins involved in DNA repair.

In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.In eukaryotes, mismatch repair plays a critical role in mutation avoidance and is carried out by the MutSLH family of proteins (for reviews, see references 13, 36, and 40). During vegetative growth, these proteins recognize and bind DNA mispairs that result primarily from replication errors or DNA damage. In Escherichia coli, MutS binding to DNA mispairs results in the recruitment of MutL, a matchmaker protein that functions in postreplicative mismatch repair by interacting with both the MutH endonuclease and UvrD helicase (22, 23). These interactions coordinate mispair recognition with DNA strand-specific signals so that mispairs are removed via excision and resynthesis steps that occur on the newly replicated strand.Eukaryotes contain multiple MutS (Msh) and MutL (Mlh) homologs, with six Msh and four Mlh homologs present in Saccharomyces cerevisiae (36). Genetic and biochemical studies have shown that the eukaryotic homologs display specialized functions with respect to the types of DNA substrates on which they act (10, 36, 40). In S. cerevisiae, the Mlh proteins form heterodimers (Mlh1p-Pms1p, Mlh1p-Mlh3p, and Mlh1p-Mlh2p) that display unique functions. Mlh1p is considered a central member of this group because heterodimers have not been identified among the other members (45, 70). The Mlh1p-Pms1p complex plays a major role in postreplicative mismatch repair, while the other two Mlh complexes appear to be redundant with Mlh1p-P...

The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser 367 , Ser 1893 , and Ser 1981 ) and acetylation on Lys 3016 . We now describe the identification of a new ATM phosphorylation site, Thr(P) 1885 and an additional autophosphorylation site, Ser(P) 2996 , that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser 367 and Ser 2996 , for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P) 1981 , ATM phosphorylated at Ser 367 and Ser 2996 localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser 367 and Ser 2996 was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo. DNA double strand breaks (DSB)4 arise during normal physiological cell processes, such as immunoglobulin and T cell receptor gene rearrangements (1), but also arise in response to exposure to a variety of DNA-damaging agents that, if left unrepaired, lead to cell death or genomic instability, cancer, and other pathologies (2). DNA DSB are repaired in mammalian cells by non-homologous end joining and homologous recombination that predominate at different stages of the cell cycle (3). The Mre11-Rad50-Nbs1 (MRN) complex is a primary sensor of DNA DSB, and there is evidence that it participates in both forms of DSB repair (4 -7). Hypomorphic mutations in members of this complex give rise to disorders characterized by sensitivity to agents that generate DNA DSB, cell cycle checkpoint defects, genome instability, and neurological abnormalities (8 -10). These syndromes overlap in their clinical and cellular phenotypes with ataxia-telangiectasia (A-T) defective in ATM kinase (11).ATM is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family of proteins and is primarily activated by DNA DSB to signal to both the DNA repair machinery and the cell cycle checkpoints (12, 13). Considerable progress has been made in understanding the mechanism of ATM activation (14 -17). Although the nature of the initial stimulus for activation remains undefined, it seems likely that relaxation of chromatin structure is suffi...