A single-molecule approach to unravel the molecular mechanism of the action of Deinococcus radiodurans RecD2 and its interaction with SSB and RecA in DNA repair.

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. They are responsible for resolving double-stranded DNA (dsDNA) into single-strands, which is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions to the organism's unusually high tolerance to gamma radiation and hydrogen peroxide. Although the results from X-ray Crystallography studies have revealed the structural characteristics of the protein, direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5' terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.