The broad long-term objective of this proposal is to elucidate the molecular basis of the Benzofalpyrene (B[a]P)-induced S-phase checkpoint. B[a]P is an abundant and ubiquitous environmental carcinogen that is metabolized intracellularly to generate Benzo[a]pyrene Di-hydrodiol-Epoxide (BPDE). BPDE-induced DMA damage triggers S-phase checkpoint signaling pathways that coordinate replication and repair of the genome. However, the molecular mechanism(s) by which checkpoint signaling regulates DNA replication are incompletely understood. Inaccurate repair and replication of damaged DNA can result in genomic instability, a hallmark of cancer cells. The BPDE-induced S-phase checkpoint is likely to help maintain genomic stability and prevent cancer after B[a]P exposure. This application will investigate the mechanisms by which S-phase checkpoints are activated by DNA damage, and the Trans-Lesion Synthesis (TLS)-mediated mechanisms that enable recovery from the checkpoint. Our studies indicate that Cdc45 (a DNA replication factor) and DNA polymerase Pol kappa (Pol?) are involved in S-phase checkpoint activation and recovery respectively. We have also shown that Pol? is regulated by the E3 ubiquitin ligase Rad18.
The Specific Aims of this proposal are: (1) To test the hypothesis that Cdc45 is a target of the BPDE-induced S- phase checkpoint. (2) To elucidate the role of Rad18 in recovery from the BPDE-induced S-phase checkpoint. (3) To elucidate the role of the Pol?-interacting DNA polymerase Rev1 in checkpoint recovery.
Aims 1 and 2 will identify and mutate sites of Cdc45 ubiquitination (Aim 1) and Rad18 phosphorylation (Aim 2). Then we will study the regulation of ubiquitination and phosphorylation-resistant mutants by checkpoint signaling. These experiments will test the significance of Cdc45 ubiquitination and Rad18 phosphorylation in relation to the S-phase checkpoint.
In Aim 3 we will determine the effect of ablating Rev1 (which interacts physically and genetically with Pol?) on Pol? regulation. These studies will determine how interactions between Pol? and Rev1 regulate the S-phase checkpoint. Together, these experiments will provide a novel paradigm for the mechanisms by which the DNA replication machinery is coordinated with DNA repair proteins in response to B[a]P (and .possibly other genotoxins) to maintain genomic stability. Results of our studies might help identify individuals that are at high-risk for environmental B[a]P-induced disease. Moreover, our studies could help identify novel drug targets for cancer therapy: Similar to B[a]P, many chemotherapies are genotoxic and activate checkpoint pathways. We have shown that Pol? or Rad18- deficiency sensitizes cells to B[a]P-induced death. Potentially, small molecules that target Rad18, Pol?, or other TLS enzymes could sensitize cancer cells to killing by chemotherapeutic agents.
Showing the most recent 10 out of 24 publications
