BRCA1 immunostaining reveals discrete, nuclear foci during S phase of the cell cycle. Human Rad51, a homolog of bacterial RecA, behaves similarly. The two proteins were found to colocalize in vivo and to coimmunoprecipitate. BRCA1 residues 758-1064 alone formed Rad51-containing complexes in vitro. Rad51 is also specifically associated with developing synaptonemal complexes in meiotic cells, and BRCA1 and Rad51 were both detected on asynapsed (axial) elements of human synaptonemal complexes. These findings suggest a functional interaction between BRCA1 and Rad51 in the meiotic and mitotic cell cycles, which, in turn, suggests a role for BRCA1 in the control of recombination and of genome integrity.

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

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A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia-and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are-300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination. [ Mitotic cells guard against genetic instability and increase their survival following DNA damage both by direct DNA repair mechanisms and by delaying progression through the cell cycle. Depending on the position of the cell within the cell cycle at the time of irradiation, DNA damage in mammalian cells can effect specific checkpoints that prevent passage from G 1 to S phase, progression through S phase, or passage from G 2 into mitosis. These checkpoints are thought to prevent dele-terious events such as replication of damaged DNA or the segregation of fragmented chromosomes during mi-tosis (Hartwell and Kastan 1994) and therefore assure the accurate transmission of the genetic material.

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.C ancer cells differ from their normal cellular counterparts in many important characteristics, including loss of differentiation, increased genomic instability, and decreased drug sensitivity. Not surprisingly, genetic alterations occur in most, if not all cancer cells, and are thought to lie at the heart of these phenotypic alterations. Furthermore, molecular analysis of individual tumors often reveals multiple genetic changes, including chromosomal translocations, deletions, insertions, gene amplifications, and point mutations. Recent surveys have identified more than 2,000 recurrent chromosomal aberrations among different neoplastic disorders (1, 2). However, the molecular and phenotypic alterations that are associated with the majority of these chromosomal changes remain undefined. The results described in this report characterize a new type of chromosomal abnormality that occurs with certain chromosome rearrangements, and is associated with abnormal chromosome replication timing, abnormal mitotic chromosome condensation, and considerable chromosomal instability. MethodsCells. C2C12, CRL-5845, CRL-5824, HTB-81, HTB-118, WERI-RB1, and HELA cells were from the American Type Culture Collection. RH30 cells were provided by P. Houghton (St. Jude Children's Hospital, Memphis, TN). All cell lines were grown in DMEM supplemented with 10% FBS (HyClone). CRL-5845 and RH30 cells were stably transfected with pRSVNEO by electroporation (300 volts, 950 F in PBS; Bio-Rad), and Ϸ2,000 clones were pooled and expanded for use as donors in microcell fusions.Microcell Mediated Chromosome Transfer. Donor cells were micronucleated by adding 10.0 g of colcemid per ml in DMEM plus 15% calf serum for 48 h. The micronucleate cell populations were enucleated by centrifugation in the presence of 5 g of cytochalasin B (Sigma) per ml, and the isolated microcells were fused to C2C12 recipients as described (3, 4). Microcell hybrids were isolated by using cloning cylinders after 3-4 weeks of selection in medium containing 500 g of Geneticin (GIBCO) per ml.Fluorescent in Situ Hybridization. Chromosome preparations from primary tumors were harveste...