The multiple enzymatic activities of DNA polymerases must be carefully orchestrated and regulated to ensure accurate and efficient DNA synthesis during DNA replication and repair, while preventing the formation of mutagenic or unstable DNA intermediates. There is a fundamental gap in understanding functional coordination, which exists across the entire DNA polymerase family. The long-term goal is to understand in molecular detail how DNA polymerases function during DNA replication and repair. The objective of this application, which is the next step in the pursuit of this goal, is to determine ow the multiple enzymatic activities of E. coli DNA polymerase I (Pol I) are coordinated and regulated. Pol I is a readily manipulated model enzyme that exemplifies the essential features of multifunctional DNA polymerases from more complex organisms. The central hypothesis is that functional coordination is linked to the conformational dynamics of Pol I. In particular, it is proposed that the flexibly tethered 5'nuclease domain, through its ability to dynamically shift position, both orchestrates and enhances the different enzymatic activities of Pol I. This hypothesis has been formulated on the basis of preliminary data from the applicant's laboratory. The rationale for the proposed research is that once the conformational states of the 5'nuclease domain and its influence on the other domains are known, it will be possible to understand how Pol I coordinates it's different activities and why this coordination sometimes fails, leading to mutations, strand breaks or other deleterious outcomes. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: (1) Dissect the nucleotide incorporation cycle of Pol I and reveal the role of the 5'nuclease domain. (2) Dissect the 3'-5'exonuclease pathway of Pol I and reveal the role of the 5'nuclease domain. (3) Determine how the 5'nuclease activity is physically coordinated with the other enzymatic activities of Pol I. A range of novel single- molecule fluorescence spectroscopic methods will be developed in order to visualize conformational changes that mediate functional coordination in the Pol I enzyme, and the single-molecule data will be corroborated in parallel biochemical experiments. The approach is innovative, because it will employ a fundamentally new experimental approach, based on direct visualization of enzyme conformational changes within a single polymerase molecule as it switches between different modes of activity during a single encounter with a DNA substrate. The proposed research is significant, because it will establish an unprecedented level of understanding of how the different enzymatic activities of a multifunctional DNA polymerase work together during DNA replication and repair.

Public Health Relevance

Improper functioning of DNA polymerases during DNA replication and repair can lead to premature aging, neurodegeneration and cancer. This project will advance our understanding of the mechanisms that orchestrate and regulate the multiple enzymatic activities of DNA polymerases, and why these mechanisms sometimes fail, leading to the formation of mutagenic or unstable DNA intermediates. The innovative single- molecule methods developed in this project will open a new window into the inner workings of a model multifunctional DNA polymerase and establish a paradigm of functional coordination that will be broadly applicable across the entire DNA polymerase family.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-P (02))
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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
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
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
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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