The goal of this research proposal is to provide insight into the biochemical mechanisms of DNA helicases at the single molecule level. To achieve this goal, this proposal is subdivided into three primary questions. By what mechanism do structurally distinct helicases facilitate processing of complex DNA substrates? How do the various helicase motifs achieve translocation and strand separation? What are the forces exerted by DNA motors as they proceed to translocate and unwind DNA? For the majority of experiments, single molecule techniques will be used to answer these three questions. To provide answers to the first two questions Dr. Bianco will combine optical tweezers and fluorescence microscopy to directly visualize these dynamic nanomachines in motion. The experiments designed to address question three will take advantage of atomic force microscopy to measure the forces in operation for DNA helicases during translocation and DNA unwinding. Dr. Bianco will use these single molecule techniques to study four DNA motor proteins that have been selected to provide both unique and complementary information on the details and nuances of the dynamics of motion of these enzymes. These Escherichia coli enzymes are the DNA helicases RecBCD, RecG, RuvAB and the type I restriction enzyme EcoR124I. It is anticipated that direct observation of these nanomachines in real time will provide novel insights into the biochemical mechanism of DNA helicases, a class of nucleic acid motors that are of fundamental importance to DNA metabolism. In addition, a more detailed understanding of these proteins will contribute to a general appreciation of the molecular events responsible for aberrant DNA metabolic processes. The importance of studying DNA helicases is emphasized by evidence demonstrating that the genetic defects leading to Bloom's syndrome, Cockayne's syndrome, Werner syndrome, and Xeroderma pigmentosum, have all been identified as mutations in DNA helicases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066831-04
Application #
7002229
Study Section
Biochemistry Study Section (BIO)
Program Officer
Lewis, Catherine D
Project Start
2003-01-01
Project End
2007-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
4
Fiscal Year
2006
Total Cost
$271,205
Indirect Cost
Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
YĆ¼ksel, Deniz; Bianco, Piero R; Kumar, Krishna (2016) De novo design of protein mimics of B-DNA. Mol Biosyst 12:169-77
Bianco, Piero R; Webb, Martin R (2012) Helicase unwinding: active or merely perfect? J Mol Biol 420:139-40
Bianco, Piero R; Stanenas, Adam J; Liu, Juan et al. (2012) Fluorescent single-stranded DNA-binding proteins enable in vitro and in vivo studies. Methods Mol Biol 922:235-44
Bianco, Piero R; Xu, Cuiling; Chi, Min (2009) Type I restriction endonucleases are true catalytic enzymes. Nucleic Acids Res 37:3377-90
Buss, Jackson A; Kimura, Yuji; Bianco, Piero R (2008) RecG interacts directly with SSB: implications for stalled replication fork regression. Nucleic Acids Res 36:7029-42
Brewer, Laurence R; Bianco, Piero R (2008) Laminar flow cells for single-molecule studies of DNA-protein interactions. Nat Methods 5:517-25
Bianco, Piero R; Bradfield, Justin J; Castanza, Lauren R et al. (2007) Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis. J Mol Biol 374:618-40
Slocum, Stephen L; Buss, Jackson A; Kimura, Yuji et al. (2007) Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA. J Mol Biol 367:647-64
Anand, Syam P; Zheng, Haocheng; Bianco, Piero R et al. (2007) DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange. J Bacteriol 189:4502-9
Kimura, Yuji; Bianco, Piero R (2006) Single molecule studies of DNA binding proteins using optical tweezers. Analyst 131:868-74

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