The long-term goal of this project is to elucidate the molecular basis for DNA polymerase fidelity and the role of protein conformational dynamics in polymerase function. Accurate nucleotide selection and proofreading by DNA polymerases are essential for proper cellular physiology and the faithful transmission of genetic information during cell division. This project will focus on dynamic conformational changes of the polymerase-DNA complex that are linked to the nucleotide incorporation and 32-52 exonucleolytic proofreading activities. We will study the high fidelity Klenow fragment polymerase, which provides a well-characterized model system.
The specific aims are: (1) Develop single-molecule fluorescence methods to monitor the conformational dynamics of DNA polymerases. (2) Establish how functionally important conformational changes of DNA polymerase are linked to selection of a correct incoming nucleotide substrate. (3) Detect DNA polymerase activity at the single- molecule level in real-time. (4) Discover how a DNA primer/template travels between the separate polymerase and 32-52 exonuclease sites during proofreading. (5) Establish how the polymerase and exonuclease activities are physically coordinated to achieve high fidelity DNA replication. Novel single- pair FRET systems will be established to monitor enzyme conformational changes during nucleotide incorporation and translocation of DNA during proofreading. These new tools will open a unique window into the inner workings of DNA polymerase enzymes and will be broadly applicable to other protein-DNA complexes.

Public Health Relevance

Accurate nucleotide selection and proofreading by DNA polymerases are essential for the faithful transmission of genetic information during cell division and other fundamental cellular processes such as DNA repair. Moreover, specialized Y-family polymerases play an essential role in the avoidance of mutagenic changes arising from exposure to DNA damaging agents, such as sunlight and small molecule carcinogens. Improper polymerase function can cause a range of disorders, including cancer. Hence, studies of the basic mechanisms of DNA polymerases are relevant to understanding the causes of human genetic diseases and cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Lewis, Catherine D
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Scripps Research Institute
La Jolla
United States
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Lavergne, Thomas; Lamichhane, Rajan; Malyshev, Denis A et al. (2016) FRET Characterization of Complex Conformational Changes in a Large 16S Ribosomal RNA Fragment Site-Specifically Labeled Using Unnatural Base Pairs. ACS Chem Biol 11:1347-53
Millar, David P; Trewhella, Jill (2014) Editorial overview--New frontiers of biophysical methods: tools for structural biology and beyond. Curr Opin Struct Biol 28:viii-x
Lamichhane, Rajan; Berezhna, Svitlana Y; Gill, Joshua P et al. (2013) Dynamics of site switching in DNA polymerase. J Am Chem Soc 135:4735-42
Ridgeway, William K; Millar, David P; Williamson, James R (2013) Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy. Comput Phys Commun 184:1322-1332
Berezhna, Svitlana Y; Gill, Joshua P; Lamichhane, Rajan et al. (2012) Single-molecule Forster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I. J Am Chem Soc 134:11261-8
Ridgeway, William K; Millar, David P; Williamson, James R (2012) Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy. Proc Natl Acad Sci U S A 109:13614-9
Ridgeway, William K; Millar, David P; Williamson, James R (2012) The spectroscopic basis of fluorescence triple correlation spectroscopy. J Phys Chem B 116:1908-19
Gill, Joshua P; Wang, Jun; Millar, David P (2011) DNA polymerase activity at the single-molecule level. Biochem Soc Trans 39:595-9
Tahmassebi, Deborah C; Millar, David P (2009) Fluorophore-quencher pair for monitoring protein motion. Biochem Biophys Res Commun 380:277-80
Stengel, Gudrun; Gill, Joshua P; Sandin, Peter et al. (2007) Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Biochemistry 46:12289-97

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