Large domain movements upon UvrD dimerization and helicase activation

Abstract

UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

“…The SF1 enzymes, Rep, UvrD, and PcrA are rapid, directional, and processive ssDNA translocases as monomers, but have no detectable helicase activity unless the DNA is under tension . The helicase activity of these enzymes needs to be activated either by dimerization, interaction with an accessory protein, removal of the auto‐inhibitory 2B sub‐domain, or crosslinking of the 2B sub‐domain into its closed conformation . In fact, it was demonstrated 25 years ago that a dimeric Rep helicase is able to unwind a short duplex DNA beyond a 5′‐5′ RP linkage in the 3′ ssDNA tail loading site, thus showing a similar ability to that of RecBCD .…”

“…In spite of much research, the mechanisms by which helicases unwind duplex DNA are not understood. It is clear from studies of the SF1 helicases, Rep, UvrD, and PcrA, that ATP‐dependent single stranded DNA translocase activity is not sufficient to yield helicase activity . Here we discuss recent evidence suggesting that processive DNA unwinding by the E. coli RecBCD helicase is a two‐step process involving ATP‐independent DNA melting due to the favorable free energy of the protein–DNA interaction, followed by ATP‐dependent ssDNA translocation.…”

Processivity, velocity and universal characteristics of nucleic acid unwinding by helicases

“…The "processive, zipper-like" ds unwinding activity was first demonstrated by Abdel-Monem and colleagues in E. Coli based DNA helicase in 1976 [10]. Interestingly however, most helicases cannot unwind double strands of nucleic acids by themselves, and require either oligomerization or assistance from other partner proteins to increase the efficiency of processive unwinding [11][12][13]. The fundamental principles governing this collaborative improvement of unwinding efficiency have not been fully elucidated.…”

“…Universal nature of force response of helicase processivity, oligomerization and partner proteins: Helicases often cannot unwind double strand nucleic acids by themselves, but require partner proteins like single strand binding proteins or oligomerization to increase the efficiency [11][12][13]. We use efficiency to mean that the helicase is highly processive.…”

“…10, 2018; Similar effects may be achieved by oligomerization of helicase monomers. A recent study on the UvrD helicase showed that dimerization leads to a closed conformation of the 2B subdomain of the leading UvrD monomer [13], a conformation that contacts the junction duplex and presumably destabilizes it [53,54]. This implies the possibility that a universal underlying principle governs the response of helicases to force, partner proteins as well as oligomerization.…”