Two Structurally Different Families of DNA Base Excision Repair (BER) Proteins Diffuse Along DNA to Find Intrahelical Lesions

Abstract

The mechanism of helicase translocation on DNA remains controversial and the translocase activity driving their non-canonical functions such as protein displacement is poorly understood. Here, we used single molecule fluorescence assays to study a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, and discovered a progressive looping of ssDNA that is tightly coupled to PcrA translocation on DNA. Variance analysis of hundreds of looping events by a single protein demonstrated that PcrA translocates on ssDNA in uniform steps of 1 nt, reconciling discrepancies in previous structural and biochemical studies. On the forked DNA, rather than acting on the leading strand to unwind the duplex, PcrA anchored itself to the duplex junction and reeled in the lagging strand using its 3'-5' translocation activity. PcrA maintained the open conformation, not the closed conformation observed in crystallographic analysis, during looping-coupled translcation. This activity could rapidly dismantle a preformed RecA filament even at 1nM PcrA, suggesting that the translocation activity and structure-specific DNA binding are responsible for removal of potentially deleterious recombination intermediates.