Genetic Analysis of a Mammalian Chromosomal Origin of Replication
Abstract
The purpose of this study was to begin to understand the mechanism of replication initiation in mammalian cells in order to gain insight into how misregulation of initiation may lead to cancer progression. We have shown that a 5.8 kb DNA fragment containing the initiation region (IR) DHFR ori-beta is active at ectopic chromosomal locations in hamster cells and that deletion of three specific elements in ori-beta reduced initiation activity. Further characterization of these elements showed that an AT-rich element is required for efficient ori-beta activity, and that a homologous region of the human lamin B2 origin can substitute for this element. In addition, the 5.8 kb ori-beta DNA fragment is sufficient to direct replication initiation in two human cancer cell lines, suggesting a possible conservation of the mechanisms of replication initiation in mammalian cells. In order to begin to understand the relationship of origin activity and initiator protein binding, we assessed protein binding to ectopic ori-beta in a human cancer cell line. We found that initiation proteins localized to the replication start site of ori-beta and deletion of the AT-rich element modulated protein binding. Taken together, these results suggest a potential relationship between ORC binding and origin selection and activation.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2002
- Accession Number
- ADA408698
Entities
People
- Amy L. Altman
Organizations
- Vanderbilt University