A variety of endogenous and environmental DNA damaging agents pose a constant threat to DNA. Ultraviolet (UV) light from the sun is a common source of DNA damage in organisms, including humans. Exposure to UV light causes lesions in DNA that can impede DNA polymerases resulting in stalled replication forks. Failure to re-start stalled replication forks can have serious effects on cells, including interruptions in the cell cycle and chromosome breakage. Thus, UV-induced DNA lesions can destabilize the genome, increasing the chance of mutations. Such genome instability can lead to cancer. One way cells minimize the deleterious effects of damage that blocks DNA replication is through use of a specialized class of DNA polymerases that can replicate across the lesions in a process called translesion synthesis. DNA polymerase 7 (C. elegans POLH-1) is especially important for translesion synthesis past UV-induced lesions. Polymerase 7 and other translesion polymerases have high error rates when replicating undamaged template. Thus, their access to DNA replication forks must be tightly regulated to prevent mutagenesis. How translesion polymerases are removed from replication forks after TLS, to minimize their engagement with undamaged DNA, is not currently known. Recent work from our laboratory has elucidated a possible mechanism for how this might occur. According to our model, when POLH1-1 is recruited to replication forks, it is SUMOylated by GEI-17 E3 SUMO ligase, and stabilized long enough to perform translesion synthesis. The mechanism by which GEI-17 senses DNA damage to SUMOylate POLH-1 is not known. After translesion synthesis, POLH-1 is ubiquitinated by CDT-2 ubiquitin ligase, targeting it for degradation. Thus, we hypothesize that CDT-2-mediated degradation of POLH-1 limits its access to undamaged template, controlling POLH-1's mutagenic capacity. The experiments described in this proposal will address two outstanding questions related to this hypothesis.
In Aim 1, I will use genetic and biochemical approaches to clarify the mechanism for how GEI-17 is recruited to sites of DNA damage to SUMOylate POLH-1.
In Aim 2, I will use high throughput DNA deep sequencing to examine whether CDT-2 controls mutagenesis.
The variant form of the genetic disorder Xeroderma pigmentosum (XP-V) is caused by loss of polymerase 7 function. XP-V patients are hypersensitive to sunlight and have an increased predisposition for skin cancer. This pathology suggests that human DNA polymerase 7 limits the carcinogenic potential of UV light. A more complete understanding of polymerase 7 function, coming from my studies of POLH-1, could contribute to the eventual development of advanced cancer treatment therapies.
The experiments in this proposal will explore the regulation of DNA polymerase POLH-1 in Caenorhabditis elegans. Homologs of POLH-1 (DNA polymerase 7 in humans) minimizes mutations caused by UV light, thus limiting the development of cancer. Patients with a defect in the function of polymerase 7 are highly sensitive to UV light and have a predisposition for cancer. Thus, a detailed understanding of the molecular activities of polymerase 7 and its regulation may contribute to the creation of new cancer treatments.