There is a fundamental gap in understanding how stalled DNA replication forks are regressed and subsequently restored. Continued existence of this gap represents an important problem because, until it is filled, a complete and clear understanding of the mechanism of stalled fork reactivation will be lacking. This understanding is crucial as defects in these repair mechanisms in higher organisms lead to the accumulation of mutations leading to cancer, and the proposed studies are therefore directly relevant to human disease. Consequently, the long term goal is to understand the mechanism of stalled DNA replication fork reactivation. The objective of this proposal is to understand the mechanisms of enzyme-catalyzed, fork regression and of the subsequent enzymatic processing events leading to restoration of a fork structure. To achieve this objective, this proposal is divided into two specific aims: 1) Determine the mechanism of processing of fork substrates by key recombination helicases;and 2) Determine the biochemical mechanisms of fork reactivation on single molecules of DNA. Under the first aim, bulk-phase biochemistry and atomic force microscopy will be used to determine how recombination helicases bind to and process substrates that mimic stalled forks and substrates that mimic regressed forks. When the proposed studies for Aim 1 are complete, a clear picture of the role of each enzyme in fork reactivation will be provided. Under the second aim, two single DNA molecule approaches will be used to produce real-time movies of the molecular events occurring at stalled DNA replication forks with high spatial and temporal resolution. At the conclusion of the proposed studies for aim 2, the first visual and real-time insight into the range of events that transpire t reactivate a stalled fork in vivo will be provided. The proposed research is innovative because of the combinatorial strategy taken, the novel single molecule approaches used and the care taken in elucidating how recombination helicases function in the presence of SSB. The proposed research is significant because it will allow for the first time, the development of clear models o the mechanistic events occurring at a stalled fork and it will provide the first visual and real- tme insight into the range of events that transpire to reactivate a stalled fork in vivo.
Understanding how stalled DNA replication forks are rescued is important to public health as defects in these repair mechanisms in higher organisms lead to the accumulation of mutations leading to cancer. The proposed research is relevant to the part of NIH's mission that pertains to fostering fundamental creative discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health.
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