How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Abstract

DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the mechanisms by which these enzymes carry out the function for which they are named. Most current views are based on inferences from crystal structures. A prominent view is that the canonical ATPase motor exerts a force on the ssDNA resulting in "pulling" the duplex across a "pin" or "wedge" in the enzyme leading to a mechanical separation of the two DNA strands. In such models, DNA base pair separation is tightly coupled to ssDNA translocation of the motors. However, recent studies of the Escherichia coli RecBCD helicase suggest an alternative model in which DNA base pair melting and ssDNA translocation occur separately. In this view, the enzyme-DNA binding free energy is used to melt multiple DNA base pairs in an ATP-independent manner, followed by ATP-dependent translocation of the canonical motors along the newly formed ssDNA tracks. Repetition of these two steps results in processive DNA unwinding. We summarize recent evidence suggesting this mechanism for RecBCD helicase action.

Synergy between RecBCD subunits is essential for efficient DNA unwinding

“…First, it was shown in the crystal structure of RecBCD that there is a ‘pin’ domain in RecC against which the fork is split enabling RecB and RecD to pull the opposite unwound ssDNA strands (Singleton et al, 2004). Second, it has been postulated that the ‘arm’ protrusion in RecB may play a direct role in destabilizing the duplex ahead of the translocating enzyme (analogous to the role suggested for auxiliary domain 2B of the PcrA helicase) (Dillingham and Kowalczykowski, 2008; Velankar et al, 1999) and a recent work showed that an arm deletion results in RecBCD’s inability to unwind DNA (Lohman and Fazio, 2018; Simon et al, 2016). In both cases, the synergy between the subunits is expressed in the separation between the domain responsible for destabilizing the fork, and the fast translocase pulling the structurally unwound strands.…”

“…As a result, one model of RecBCD’s translocation postulates that the helicase subunits pull the nascent DNA strands against the pin to catalyze unwinding. Moreover, it was proposed that there is a separation between helicase translocation and unwinding, whereby RecBCD unwinds 4–6 bp of DNA using the pin in a distinct step, and only then the helicase subunits pull the unwound DNA (Lohman and Fazio, 2018). In addition, an ‘arm’ domain of RecB interacts with the DNA 12 bps ahead (Saikrishnan et al, 2008; Singleton et al, 2004).…”

“…It was suggested to direct the DNA ends toward the helicase, acting as a guide for the duplex DNA during translocation, to mediate the large translocation step across ssDNA gaps, or to play a direct role in destabilizing the duplex ahead of the translocating enzyme (Bianco and Kowalczykowski, 2000; Dillingham and Kowalczykowski, 2008; Simon et al, 2016). Nonetheless, since the DNA encounters these two structural domains before reaching the helicase domains in RecB and RecD, it was proposed that they play roles in destabilizing the fork (Lohman and Fazio, 2018; Simon et al, 2016; Wu et al, 2012; Xie et al, 2013), but their role in the unwinding reaction is still unclear.…”

History of DNA Helicases

“…The discovery of proteins capable of ATP-dependent enzymatic unwinding of duplex DNA was first reported in 1976 by Hoffmann-Berling and colleagues at the University of Heidelberg [1,2] and Mackay and Linn at the University of California, Berkeley [3]. As pointed out recently in a review by Lohman and Fazio [4], the term "helicase", referring to an ATP-dependent duplex DNA unwinding enzyme, was coined by Hoffmann-Berling in 1978 [5] and appeared in two subsequent publications in 1979 [6,7]. Even at this early stage, a suggestion was made that different helicase enzymes were unique in terms of properties and mechanism of action (e.g., processivity).…”

Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

“…In Escherichia coli and other enteric bacteria, DSB repair and recombination require RecBCD, a complex three-subunit 330 kDa enzyme with both DNA helicase and DNA nuclease activities ( 1 , 2 , 3 ). Its multiple activities are involved in the initial steps of DSB repair and recombination (Figure 1A ).…”