Genomic DNA contains information which must be faithfully maintained and precisely decoded in order for hereditary instructions to be accurately passed on and cellular components to be properly constructed. Although DNA is remarkably stable, it is nevertheless susceptible to spontaneous damage from endogenous and exogenous sources. Despite the protection provided by DNA repair pathways, some damage may evade detection and persist into S-phase. However, replicative polymerases (pols) have very stringent polymerase domains and distinct "proofreading" domains to ensure that genomic DNA is accurately copied. Consequently, damaging modifications to DNA bases, collectively referred to as base lesions, stall or completely block progression of the replication fork, causing it to collapse. Failure to restart often results in double-strand breaks which may lead to gross chromosomal rearrangements, cell-cycle arrest, and cell death. Therefore, it is often more advantageous to bypass such replicative arrests and postpone repair of the offending damage to complete the cell cycle and maintain cell survival. Such a task may be carried out by translesion DNA synthesis (TLS), a unique process by which DNA is replicated past damage without repairing it by specialized TLS pols. Characterized by a more "open" polymerase active site and the lack of proofreading activity, TLS pols are able to stably incorporate dNTPs opposite damaged templates in a relatively error-free manner, allowing replication to proceed. However, each of the 7 or so human TLS pols replicates each base lesion with varying levels of accuracy. Therefore, selection of the inappropriate TLS pol for a given lesion may result in erroneous replication of the damaged DNA. Thus, TLS must be tightly regulated to minimize replication errors. Failure to do so may lead to the buildup of mutations and ultimately cancer. Our long term goal is to understand the mechanisms that control efficient TLS in human cells in the hopes of identifying malfunctions which promote mutation and contribute to the onset of cancer. Towards this aim, we have begun to investigate how human replicative and TLS pols exchange during DNA replication. Upon encountering damaged DNA, a replicative DNA pol must be switched out for a TLS pol in order for replication to proceed. To limit the input of less-stringent TLS pols and resume high-fidelity replication following TLS, this switch must then be reversed. Using ensemble and single-molecule kinetic approaches, including FRET and state-of-the-art zero mode waveguide technology for visualizing single molecules at biologically relevant concentrations, we will monitor these switching events in vitro to determine how they are coordinated and how the appropriate TLS pol is selected for efficient TLS across a given lesion. It is the goal of this proposal to determine how pol switching is controlled during TLS and how this control ensures efficient and specific pol switching to limit replication errors. Such knowledge will aid in identifying malfunctions which lead to erroneous pol switching during TLS and contribute to the onset of cancer by promoting mutation.
The prevalence of cancer is astounding, and, according to the National Cancer Institute, approximately forty percent of all men and women in the United States alone will develop cancer during their lifetimes. Furthermore, translesion DNA synthesis often affords cancer cells the ability to become resistant to many commonly used chemotherapeutic agents. By elucidating the mechanisms underlying translesion DNA synthesis, this research will enhance our understanding of the causes of cancer and may ultimately lead to the development of novel methods of prevention and treatment, both of which are critical to eradicating this disease and vitally important to public health.
|Hedglin, Mark; Perumal, Senthil K; Hu, Zhenxin et al. (2013) Stepwise assembly of the human replicative polymerase holoenzyme. Elife 2:e00278|
|Hedglin, Mark; Kumar, Ravindra; Benkovic, Stephen J (2013) Replication clamps and clamp loaders. Cold Spring Harb Perspect Biol 5:a010165|
|Hedglin, Mark; Kumar, Ravindra; Benkovic, Stephen J (2013) Replication clamps and clamp loaders. Cold Spring Harb Perspect Med 3:a010165|