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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM100156-04S1
Application #
9263530
Study Section
Program Officer
Reddy, Michael K
Project Start
2013-06-07
Project End
2017-02-28
Budget Start
2016-03-01
Budget End
2017-02-28
Support Year
4
Fiscal Year
2016
Total Cost
$118,093
Indirect Cost
Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
038633251
City
Amherst
State
NY
Country
United States
Zip Code
14228
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Sun, Zhiqiang; Hashemi, Mohtadin; Warren, Galina et al. (2018) Dynamics of the Interaction of RecG Protein with Stalled Replication Forks. Biochemistry 57:1967-1976
Bianco, Piero R; Pottinger, Sasheen; Tan, Hui Yin et al. (2017) The IDL of E. coli SSB links ssDNA and protein binding by mediating protein-protein interactions. Protein Sci 26:227-241
Pan, Yangang; Sun, Zhiqiang; Maiti, Atanu et al. (2017) Nanoscale Characterization of Interaction of APOBEC3G with RNA. Biochemistry 56:1473-1481
Bianco, Piero R; Lyubchenko, Yuri L (2017) SSB and the RecG DNA helicase: an intimate association to rescue a stalled replication fork. Protein Sci 26:638-649
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Wang, Zhaojun; Cai, Yanan; Liang, Yansheng et al. (2017) Single shot, three-dimensional fluorescence microscopy with a spatially rotating point spread function. Biomed Opt Express 8:5493-5506
Tan, Hui Yin; Wilczek, Luke A; Pottinger, Sasheen et al. (2017) The intrinsically disordered linker of E. coli SSB is critical for the release from single-stranded DNA. Protein Sci 26:700-717
Yu, Cong; Tan, Hui Yin; Choi, Meerim et al. (2016) SSB binds to the RecG and PriA helicases in vivo in the absence of DNA. Genes Cells 21:163-84
Zhang, Yuliang; Hashemi, Mohtadin; Lv, Zhengjian et al. (2016) Self-assembly of the full-length amyloid A?42 protein in dimers. Nanoscale 8:18928-18937

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