Abstract
The mechanisms by which metazoan origins of DNA replication are defined, regulated, and influenced by chromosomal events remain poorly understood. To gain insights into these mechanisms, we developed a systematic approach using a Drosophila high-resolution genomic microarray to determine replication timing, identify replication origins, and map protein-binding sites along a chromosome arm. We identify a defined temporal pattern of replication that correlates with the density of active transcription. These data indicate that the influence of transcription status on replication timing is exerted over large domains (>100 kb) rather than at the level of individual genes. We identify 62 early activating replication origins across the chromosome by mapping sites of nucleotide incorporation during hydroxyurea arrest. Using genome-wide location analysis, we demonstrate that the origin recognition complex (ORC) is localized to specific chromosomal sites, many of which coincide with early activating origins. The molecular attributes of ORC-binding sites include increased AT-content and association with a subset of RNA Pol II-binding sites. Based on these findings, we suggest that the distribution of transcription along the chromosome acts locally to influence origin selection and globally to regulate origin activation.