Investigation of Intradomain Motions of a Y-Family DNA Polymerase during Substrate Binding and Catalysis.

DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y-family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.