During the DNA synthesis (S) phase of the cell-cycle, origins of DNA replication fire at discrete sites on human chromosomes. The replication forks that initiate at these origins move bi-directionally and terminate when they encounter forks coming from the opposite direction. The identities of the origins and termination zones are poorly understood in human chromosomes. In addition, all origins do not fire synchronously at the onset of S phase. Some origins fire early in S phase while others fire late in S phase. Not much is know about the distribution of such early and late firing origins on human chromosomes and their location relative to actively transcribed genes. In this project, using methods validated in the laboratory, DNA replicated at specific times in S phase will be purified from HeLa cells synchronously released from the G1-S boundary. These timed-replication products will be used as probes on microarrays of DNA fragments representing the 30 mB of the human genome that is the target of this RFA. A plot of the time of replication versus physical location of the DNA markers will allow the identification of origins, both early and late, because origin specific segments will replicate earlier than adjoining segments. The plot will also reveal where replication forks moving in opposite directions meet (or terminate). The slope of the line on either side of putative origins will be a measure of the speed of replication fork movement. A subset of the origin containing fragments will be confirmed to be bona-fide origins by neutral/neutral 2D gel electrophoresis of replication intermediates harvested from cells at the time in S phase when these origins are expected to initiate. This analysis will allow the distinction of single site origins versus initiation zones containing a cluster of origins. Collectively the results will identify important replication elements: origins, initiation zones and termination sites. In addition, they will identify sites in the chromosomes with replication pause sites and/or zones where replication profiles change abruptly.
