Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.

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The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCIDresponsible gene. We isolated an ORF of the murine DNAPKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.

NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N -nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95 ؉/؉ ) or heterozygously inactivated (Np95 ؉/؊ ) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to xrays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95 ؉/؊ cells, although slightly below that of wild type (Np95 ؉/؉ ) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95 ؉/؉ cells or Np95 ؉/؊ cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability.Entry into and progression through the mammalian cell cycle are highly regulated processes that involve a number of positively and negatively acting proteins at the molecular level. When growing cells are exposed to DNA damage or DNA replication blocks, they arrest their cell cycle processes, apparently to allow time for repair to be completed satisfactorily. This arrest is part of a "checkpoint" function that monitors the physical state of DNA at different stages of the cycle. Cells that have lost a checkpoint control may be as DNA damage-sensitive as cells that have lost DNA repair capability. Many genes are involved in controlling the cell cycle and determining checkpoints (1-3). Furthermore, a possible link between checkpoint failure, hypersensitivity to DNA damage or replication blockage, and genomic instability has provided an important insight into processes contributing to the cellular dysfunction that leads to cancer (4).We previously produced a monoclonal antibody, Th-10a mAb, 1 that recognizes a 95-kDa mouse nuclear protein (NP95). NP95 was detected by the Th-10a mAb, specifically in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells showed a constantly high level for NP95 accumulation irrespective of cell stages during the cell cycle (5). By immunoscreening a gt-11 cDNA expression library with the Th-10a mAb, we isolated the cDNA encoding NP95 (6). Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame consisting of 782 amino acids. The open reading frame contains an unusual N-terminal domain that bears a striking resemblance to ubiqutin, a leucine zipper motif, a zinc ...

We previously obtained a monoclonal antibody (Th-10a mAb) that recognizes a single 95-kDa mouse nuclear protein (NP95). Immunostaining analyses revealed that the NP95 was specifically stained in the S phase of normal mouse thymocytes. In contrast, mouse T cell lymphoma cells exhibited a constantly high level of NP95 accumulation irrespective of cell stages during the cell cycle. In the present study, we isolated the cDNA encoding the NP95 from a lambdagt-11 cDNA expression library, using the Th-10a mAb. Sequencing of the whole 3.5-kb cDNA revealed that NP95 is a novel nuclear protein with an open reading frame (ORF) consisting of 782 amino acids. The ORF contains a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A/E-cdk2 phosphorylation site, and the retinoblastoma protein (RB)-binding motif "IXCXE". The chromosomal location of Np95 gene was determined by fluorescence in situ hybridization. Np95 gene locates on mouse Chromosome (Chr) 17DE1.1. and rat Chr 9q11.2-q12.1. Np95 was strongly expressed in the testis, spleen, thymus, and lung tissues, but not in the brain, liver, or skeletal muscles. These results collectively implicate this novel nuclear protein in cell cycle progression and/or DNA replication.