Principal Investigator Stephen Bell
We have extended our studies to the fruit fly Drosophila melanogaster to address how origin selection and activation occur in multi-cellular organisms. Unlike the relatively well-defined mechanisms of origin selection in S. cerevisiae, origin selection in multi-cellular organisms is poorly understood. Although ORC still plays a primary role in origin selection, studies of ORC derived from multi-cellular organisms indicate that it has a limited ability to recognize specific DNA sequences. These findings suggest that additional mechanisms influence origin selection in these organisms (e.g. local chromatin structure or chromosomal events).
To investigate the control of origin selection in Drosophila, we have used a genomic microarrays to characterize the replication and transcription dynamics across a chromosome arm. Studies reveal a defined temporal pattern of replication that correlates with the density of active transcription as measured by RNA Pol II association with the DNA. Findings indicate that early-replicating domains are defined by the integration of transcriptional status of numerous adjacent genes spanning >100 kb. These data argue that the time of replication initiation is unlikely to be influenced by changes in the expression of individual genes. By combining the results of high resolution mapping of the earliest sites of DNA replication and the identification of ORC binding sites across the chromosome, we have identified 62 new Drosophila origins of replication. Findings suggest that there are multiple molecular determinants of ORC binding to these origins, including increased AT-content and association with promoter-proximal RNA Pol II binding sites. Findings have led us to propose that the distribution of transcription across the genome acts locally to influence ORC binding/origin selection and globally to modulate origin activation/replication timing. Current studies are using the origins we have identified to understand the mechanisms that control the in vivo specificity of ORC DNA association and origin selection. We are also testing the hypothesis that global, but not local, changes in chromatin structure influence replication timing.