Although great progress has been made in the past two decades in understanding the mechanism of initiation of DNA replication in eukaryotic cells, we still do not know what controls the times at which different origins fire during S phase. This is an important issue, because improperly regulated replication timing is associated with genomic instability and with cancer. Here the laboratories of Dr. Joel Huberman at Roswell Park Cancer Institute, Buffalo, NY, and of Dr. Janet Leatherwood at the State University of New York, Stony Brook, NY, propose to take advantage of the recently completed genome sequence of the model eukaryotic organism, the fission yeast Schizosaccharomyces pombe, to enable microarray-based genome-wide replication timing analyses in wild-type and mutant fission yeast cells. We shall utilize the new facility for fission yeast genome-wide microarray analyses developed by Dr. Leatherwood, and we shall also take advantage of earlier progress made by both laboratories toward understanding replication timing control. The results of the proposed studies will allow us to determine the locations of replication origins, their firing efficiencies, and the times at which they fire. By comparing the DNA sequences of early- and late-firing origins in wild-type cells, we may be able to identify sequence motifs important for replication timing control. By comparing the replication timing of each genomic region with its level of gene expression, we shall find out whether or not there are correlations between replication timing and gene expression in fission yeast. By comparing origin firing times between wild type cells and cells bearing specific mutated genes, we shall evaluate the importance of each tested gene for control of replication timing. We intend to focus on the roles of genes that control histone acetylation and genes that control S-phase checkpoint responses to DNA damage and replication stress.
