GENES THAT CONTROL GENOMIC STABILITY DURING REPLICATION

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To maintain genetic stability a cell must copy each segment of its genome once and only once per cell cycle. We are trying to learn how eukaryotic cells achieve this goal, and so have developed a screen for mutants of the yeast Saccharomyces cerevisiae that fail to obey the principle of one round of replication at each locus per cycle. This screen relies on the idea that if a cell loses the ability to recognize which sequences it has already copied it will make the mistake of over-replicating parts of its genome, but can be rescued from this fate if it is blocked from synthesizing DNA. Using this screen we have isolated a mutation that fails to honor the normal rules of S phase, and instead allows re-replication to occur before the products of synthesis have been segregated. We have named the mutation dos1-1 (DNA over-synthesis). The defect in dos1-1 cells does not appear to involve an inappropriate initiation of a new S phase, since the over-replication does not result in a doubling of the DNA content. Instead, cells appear to have re-replicated only about 50% of their DNA. Most of the excess DNA replication results from sequences distributed about the entire genome, but about 20% represents amplification of a specific region to about 50 times its normal copy number. These changes in DNA content appear to occur in the absence of cell or nuclear division. The DOS1 gene has been identified and its nucleotide sequence suggests that the gene product has an active site similar to type 1 DNA topoisomerases. Our preliminary results indicate that this homology is meaningful rather than coincident. The inability of cells to maintain genomic sequences at a constant copy number has recently been cited as a causative factor in common forms of colorectal cancer, and amplification of genes has long been associated with transformed cells in culture as well as in tumorous tissues. In fact, the loss of mechanisms that promote genomic stability has been associated with the early stages of formation of many types of carcinoma. To understand how genomes become unstable, it is necessary to know how cells normally promote stability. In this proposal, we seek to further characterize the functions of the DOS1 gene in maintaining genomic copy number control in yeast, and to explore the utility of our genetic screen for identifying other genes that function to limit replication to a single copy during S phase.