Single-Molecule FRET Studies of a Y-family Polymerase Provide New Insights into Nucleotide Binding Mechanism

Abstract

High fidelity polymerases are normally blocked by damage in the DNA template. Stalled replisomes can lead to DNA double-strand breaks or other detrimental genotoxic effects, thereby increasing genome instability. A specialized variety of DNA-polymerases is mobilized when replication across damaged bases (translesion DNA synthesis) is required. Y-family polymerases perform the majority of translesion synthesis and are generally found to be specialized for specific types of lesions. These Y-family polymerases typically present a wide active site to accommodate distorted/bulky DNA.Dpo4 is a Y-family polymerase that has been extensively characterized by ensemble experiments. However, the exact polymerization mechanism that occurs during the bypass of the DNA damage remains unclear. We have used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the interactions between DNA and Dpo4. Our data show that Dpo4 binds DNA in two different conformations that interconvert reversibly. Experiments carried out in the presence of nucleotides using Ca2+ instead of Mg2+ to prevent DNA extension suggest that one of these conformations is preordered to accept the incoming nucleotide. We were able to characterize the binding and dissociation dynamics between nucleotides and the DNA/Dpo4 complex. Our results also suggest that Dpo4 undergoes a similar conformational rearrangement upon binding a correct or incorrect (mismatched) nucleotide.