One of the cellular alterations required for development of cancer is genomic instability, a term that describes a collection of pathways all leading to increase frequency of gene mutation and altered patterns of gene expression. Among the mechanisms causing genomic instability are chromosome breakage, gene amplification, and changes in levels of gene expression. The latter are frequently associated with changes in gene methylation status. All of the mechanisms have been correlated with changes in replication timing, and it is possible that in some cases changes in replication timing may be the primary cause of these phenomena. So far it has been difficult to evaluate the role of changes in replication timing in cancer development, because so little is known about the mechanisms controlling replication timing. The experiments proposed in this application are intended to provide an entry point into understanding the mechanism of replication timing. We intend to take advantage of previous work in our laboratory and others which shows that ARS301, and 94-bp stretch of chromosomal DNA in the budding yeast, Saccharomyces cerevisiae, is capable of serving as a replication origin but differs from previously studied origins in being inherently late replicating. It is also of interest that ARS301 is an important part of a transcriptional silencer element, and transcriptionally repressed genes tend to replicate late. We intend to determine whether the late firing of ARS301 is due to the presence of late-replication-determining sequences or to the absence of early-replication determining sequences within it. In either case, we shall employ standard mutagenic techniques followed by replication timing tests to narrow down and characterize the responsible cis-acting sequences, so that they can be used to help identify the DNA-binding proteins that are responsible for controlling replication timing.
