Poster abstracts
Poster number 57 submitted by Varun Gadkari
Binding Dynamics and Nucleotide Selectivity of Dpo4 in Response to Oxidative Damage as Revealed by Single-Molecule FRET
Varun V. Gadkari (Ohio State Biochemistry Program), Austin T. Raper (Ohio State Biochemistry Program), Brian A. Maxwell (Biophysics), Ahmed S. Beydoun (The Ohio State University, Department of Physics )
Abstract:
Translesion synthesis (TLS) DNA polymerases utilize a complex, multi-step mechanism for efficient bypass of damaged DNA to rescue stalled replication forks as revealed by extensive kinetic and structural studies. Dpo4, the lone Y-family DNA polymerase in S. solfataricus, is likely tasked with TLS responsibilities in response to various lesions in vivo and has been studied extensively as a model Y-family DNA polymerase. Previous stopped-flow fluorescence and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Recently, single-molecule fluorescence spectroscopy has provided new insights into real-time conformational dynamics of DNA polymerases during substrate binding and nucleotide incorporation. We used single-molecule Förster resonance energy transfer (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 during substrate binding. Our findings demonstrate that this TLS polymerase interacts dynamically with the DNA, sampling a variety of locations and conformations as it recognizes and specifically binds the nucleic acid substrate. We observe changes in binding distributions and dissociation kinetics upon the introduction of nucleotide. Furthermore, the overall binding behavior observed on an unmodified DNA substrate is largely conserved in the presence of an oxidative lesion with a few notable caveats including an increase in complex stability and a shift in conformational distribution.
Keywords: Single Molecule FRET, Polymerase, Oxidative Damage