DESCRIPTION (APPLICANT'S ABSTRACT): Activation and regulation of DNA replication are of fundamental importance to proper growth and differentiation of cells and development of organisms. Understanding how the molecular events that control DNA replication are altered by genetic mutations, environmental chemicals or therapeutic drugs is important in the prevention and treatment of diseases such as cancer. We will continue to use the yeast, Saccharomyces cerevisiae, to investigate how DNA replication is regulated in cell chromosomes. We have identified cis-acting DNA elements that are necessary and sufficient to specify and activate a chromosomal replication origin, 0RI305. One specific aim is to complete the characterization of a DNA unwinding element (DUE) present in 0RI305 and detected in the plasmid Autonomously Replicating Sequence, ARS3O5. We have discovered that silent replication origins near a transcriptionally-inactive chromosomal locus (HML) can be activated in the chromosome. One key component of origin silencing is the proximity of the nearest active origin in the chromosome. Our preliminary studies indicate that another important component is a chromosome position effect near HML. A specific aim is to identify DNA sequences and chromosome contexts responsible for the chromosome position effect that silences replication origins. Our preliminary studies indicate that, when activated, silent origins fire late in S phase. A specific aim is to identify the chromosome context that determines late replication timing at silent origins and to identify novel DNA elements that control late replication timing. Preliminary studies show that origin silencing is linked to the cellular response to DNA damage and that mutations in certain cell-cycle checkpoint genes activate silent origins. A specific aim is to examine the role of checkpoint genes and newly identified cis-acting mutations in controlling specific protein associations involved in origin silencing and late replication timing.