Interactions between proteins and nucleic acids are of fundamental importance in replication, transcription, recombination and other processes involved in the regulation of gene activity. The broad, long term objective of this proposal is to develop a new approach for studying the structure and dynamics of DNA:protein complexes in solution. This approach is specifically directed at a large and important class of DNA-binding proteins that do not exhibit a strong sequence-specificity in their interaction with DNA, and whose complexes with DNA are difficult to study by conventional structural methods. The proposal will initially focus on DNA:polymerase complexes as models of inherently dynamic complexes.
The specific aims are: (1) Define the binding modes of DNA polymerase bound to DNA in solution during polymerization and editing of mispaired bases. (2) Estimate the separation between the polymerase and 3'-5' exonuclease (editing) active sites in the solution complex. (3) Study how the presence of mispaired bases in the DNA substrate influences the distribution of the two binding modes of the complex. (4) Determine whether there are local changes in the base-pairing of the bound DNA substrate during editing. (5) Determine how dNTP's and divalent metal ions affect the distribution. (6) Define which amino acid residues of the polymerase affect the mode of DNA binding and the dynamics of the complex. (7) Elucidate the common structural and dynamic features of a series of different DNA polymerases. The information obtained from this study is strongly health-related since it will provide insight into the molecular mechanisms used to suppress mutations during DNA replication. The experimental design is to characterize the structure and dynamics of DNA:polymerase complexes in solution using site-specific fluorescent probes attached to synthetic DNA oligomers and picosecond time-resolved fluorescence spectroscopy. Steady-state fluorescence spectroscopy will be used to measure binding properties. The DNA substrate and the protein structure will be systematically modified by oligonucleotide synthesis and site-directed mutagenesis, respectively, and the effect on the structure, dynamics and binding affinity of the complex will be determined. The approach developed here will be applied in future studies to other DNA-binding proteins.
| 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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