Accurate replication of DNA is an essential requirement of all living organisms, and errors made in copying genetic material can result in a wide range of disorders. DNA polymerases achieve high DNA replication fidelity through the concerted action of base selection during polymerization, coupled with preferential removal of misincorporated nucleotides at a remote 3' -5' exonuclease site. Although crystal structures of DNA substrates bound to a few DNA polymerases have been solved, it is not understood how polymerization and exonucleolysis are coordinated to achieve efficient and accurate replication of DNA. In addition, the mechanistic roles of the amino acid residues that interact with the DNA substrate at the polymerase and 3' - 5' exonuclease sites are generally unknown. The broad, long term objective of this proposal is to understand the physical basis for high DNA polymerase replication fidelity. The proposal will focus on the Klenow fragment of DNA polymerase I from E. coli, which has served as a model for describing the molecular basis of template-directed DNA synthesis.
The specific aims are: 1. Investigate the mechanisms responsible for melting duplex DNA and straining the resulting single-stranded DNA substrate at the 3' -5' exonuclease site. 2. Dissect the energetics of duplex DNA binding and distortion within the polymerase domain and characterize conformational changes of the enzyme-DNA complex induced by nucleotide binding. 3. Elucidate the mechanism by which the DNA primer terminus is transferred from the polymerase site to the 3' -5' exonuclease site. Time-resolved fluorescence anisotropy decay will be used to measure the distribution of dansyl-labeled DNA primer-templates between the polymerase and 3' -5' exonuclease sites. The enzyme will be judiciously modified by site-directed mutagenesis techniques in order to test specific hypotheses concerning the nature of the critical DNA-protein interactions at the polymerase and 3' -5' exonuclease site. The fluorescent DNA substrates will also be used to monitor the rate of movement of the primer terminus between the two active sites. Data on melting of specific base-pairs within critical regions of the enzyme-bound DNA will be obtained using a novel fluorescent probe. The information obtained from this study will provide insight into the molecular mechanisms used to suppress mutations during DNA replication.
| 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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