Direct Observation of the Stepping Behavior of E. Coli UvrD Helicase

Abstract

Helicases are a diverse set of enzymes that play an essential role in genome maintenance by converting chemical energy into the mechanical work of unzipping nucleic acid duplexes. E. coli UvrD is a prototypical superfamily 1 helicase involved in methyl-directed mismatch repair and nucleotide excision repair. Recent measurements have shown that it is able to both unwind and rezip the DNA duplex. Structural, ensemble kinetic, and single-molecule studies have provided insights into the unwinding mechanism of this helicase, but there has not yet been a direct observation of the individual motor steps involved in the process. Here, we use high-resolution optical tweezers to observe directly the stepping behavior of UvrD at a single-molecule level as it unwinds and rezips a DNA hairpin. Interestingly, we measure a step size of 3-4 base pairs for both unwinding and rezipping activities. Furthermore, our analysis of the stepping kinetics indicates that a single rate-limiting step governs the process, which is consistent with ensemble studies measuring an unwinding step size of 4-5 base pairs per rate-limiting step. However, previous studies have determined that 1 ATP molecule is hydrolyzed per base pair translocated, and would predict 3-4 rate-limiting steps per unwinding step. Our results therefore suggest a mechanism whereby UvrD rapidly unwinds 3-4 base pairs, and then must slowly “reset” itself in order to continue its unwinding cycle.