This application aims to achieve electronic control of DNA polymerase function on a time scale that superimposes with rates of enzyme binding and catalysis. To achieve this aim, we will monitor the interaction of individual DNA polymerases with a nanopore sensor under voltage- induced tension. We will characterize kinetic, biochemical, and structural properties of polymerase-DNA complexes captured under voltage control in a nanopore. We will optimize nanopore measurements of polymerase function at significantly higher bandwidth than is possible using conventional techniques and in a manner that permits serial analysis of thousands of individual enzymes as they process DNA. We believe the study is innovative because it will employ a recently established nanopore technique to identify and measure translocation steps during individual catalytic cycles of replication. Discrimination between polymerase-driven translocation mechanisms should be achievable. This work is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact.

Public Health Relevance

This work focuses on mechanisms of DNA replication by DNA polymerases. It is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087484-04
Application #
8510662
Study Section
Nanotechnology Study Section (NANO)
Program Officer
Preusch, Peter C
Project Start
2010-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$259,931
Indirect Cost
$88,359
Name
University of California Santa Cruz
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
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Lieberman, Kate R; Dahl, Joseph M; Wang, Hongyun (2014) Kinetic mechanism at the branchpoint between the DNA synthesis and editing pathways in individual DNA polymerase complexes. J Am Chem Soc 136:7117-31
Lieberman, Kate R; Dahl, Joseph M; Mai, Ai H et al. (2013) Kinetic mechanism of translocation and dNTP binding in individual DNA polymerase complexes. J Am Chem Soc 135:9149-55
Dahl, Joseph M; Mai, Ai H; Cherf, Gerald M et al. (2012) Direct observation of translocation in individual DNA polymerase complexes. J Biol Chem 287:13407-21
Garalde, Daniel R; Simon, Christopher A; Dahl, Joseph M et al. (2011) Distinct complexes of DNA polymerase I (Klenow fragment) for base and sugar discrimination during nucleotide substrate selection. J Biol Chem 286:14480-92
Lieberman, Kate R; Cherf, Gerald M; Doody, Michael J et al. (2010) Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J Am Chem Soc 132:17961-72