Research is conducted in collaboration with Research Assistant Professor M. K. Raghuraman (Raghu). We are studying the regulation of replication that ensures that each chromosome is duplicated in a timely and precise way. Although the chromosomes of S. cerevisiae (average size, 800 kb) are orders of magnitude smaller than those of plants and animals, they are organized for replication in much the same way: replication occurs from multiple, closely-spaced origins and different parts of a chromosome are replicated at different times during the S phase of the cell cycle. Early on, we developed 2-dimensional gel electrophoresis techniques that allow us to map specific replication origins and to determine the efficiency with which they are activated. More recently, we have developed methods and algorithms to study replication on a genome wide scale using microarrays. The combination of these techniques, along with the tractability of the yeast genome, has allowed us to pose important questions about DNA replication in eukaryotes. Due to the semi-discontinuous nature of replication, each active replication fork will necessarily generate a stretch of single stranded template on the lagging strand. In the presence of a drug (hydroxyurea) that inhibits nucleotide synthesis these single stranded regions persist and increase in length. In cells containing a rad53 checkpoint mutation, treatment with hydroxyurea causes replication forks to remain in the immediate vicinity of origins of replication. Therefore, we reasoned, if there were some way to map the locations of these single stranded regions, it would be possible to infer the locations of origins of replication. To map the locations of single stranded regions in the genome, we isolated DNA from these cells and used it as the template for in vitro synthesis using labeled nucleotides. Since we did not denature the genomic DNA, the only regions that can serve as templates for incorporation of the labeled nucleotides are exactly the regions that were single stranded in vivo. We then mapped the locations of these single stranded regions on a genome-wide scale by hybridizing the labeled DNA to microarrays. In a single experiment we were able to map the locations of all S. cerevisiae origins. This powerful technique has been applied to another yeast, S pombe, and we hope to expand our studies on origin identification in other species, including humans. We are also working to understand the role of Rad53 protein at the replication fork.