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Review
. 2018 Jun;40(6):e1800009.
doi: 10.1002/bies.201800009. Epub 2018 Mar 30.

How Does a Helicase Unwind DNA? Insights From RecBCD Helicase

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Free PMC article
Review

How Does a Helicase Unwind DNA? Insights From RecBCD Helicase

Timothy M Lohman et al. Bioessays. .
Free PMC article

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.

Keywords: DNA helicase; allostery; kinetics; mechanism; motor protein; nuclease; translocase.

Figures

Figure 1.
Figure 1.
RecBCD-DNA structure. A. Crystal structure (PDB 1W36) of RecBCD bound to a DNA duplex. RecB subunit is red, RecC subunit is blue, RecD subunit is green, and DNA is black. B. Stylized RecBCD structure with the subunit colors as in A. The positions of the RecB SF1A translocase motor, the SF1B RecD translocase motor, the RecB arm domain, the RecB nuclease domain and the RecC subunit are noted. The approximate positions of the ATP binding sites within the RecB and RecD motors are shown in white. The RecB arm domain interacts with the duplex DNA ahead of the fork. The nuclease domain is attached to the RecB motor domain via a long (~70 amino acid) linker.
Figure 2.
Figure 2.
Proposed mechanism for processive DNA unwinding by RecBCD helicase. RecBCD initially binds to the blunt end of a duplex DNA and couples its favorable free energy of binding to melt 4–6 bp of the duplex DNA in an ATP-independent manner. RecBCD then translocates along the resulting ssDNA tracks hydrolyzing 1 ATP/motor/nucleotide until it reaches the new ss/ds DNA junction. ATP binding/hydrolysis resets the enzyme so that RecBCD can melt another 4–6 bp and then translocate along the newly formed ssDNA tracks. These DNA melting and ssDNA translocation steps are repeated during processive DNA unwinding.
Figure 3.
Figure 3.
RecBCD can unwind DNA processively in the absence of ssDNA translocation by the canonical RecB and RecD motors. RecBCD can transiently unwind duplex DNA beyond the reverse polarity switches in the DNA backbone (indicated here by yellow x’s), which prevent the canonical RecB and RecD motors from translocating along the ssDNA. This activity is dependent upon the secondary translocase activity of RecBC that is fueled by the RecB ATPase activity and requires both the RecB arm domain and the RecB nuclease domain, but not nuclease activity. It is possible that the RecB arm act as a dsDNA translocase, pulling the dsDNA towards RecBCD, resulting in the formation of ssDNA loops.

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