Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.DOI: http://dx.doi.org/10.7554/eLife.00334.001

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-doublestranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.The function of many multisubunit DNA repair complexes requires activity of DNA helicases, which are ubiquitous, highly diverse molecular motors that convert the chemical energy of ATP binding and hydrolysis into mechanical work of unidirectional translocation along the DNA lattice (reviewed in Refs. 1, 2). Unidirectional translocation of a helicase may be coupled to other thermodynamically unfavorable processes, including separation of the nucleic acid duplexes and disassembly of protein-nucleic acid complexes. Coupling of ATP hydrolysis to translocation is achieved through the set of so-called "helicase signature motifs" that define the motor core of the enzyme (3, 4). The motor cores of numerous helicases are structurally and mechanistically similar, yet these enzymes display remarkable functional diversity (for review see Ref. 4). It has become clear in recent years that such diversity may be achieved in trans through utilization of specific processivity factors or in cis by incorporating additional structural features that direct interaction with nucleic acids, duplex destabilization, and strand separation activities.The Rad3 (XPD) 2 helica...

SUMMARY An encounter between a DNA translocating enzyme and a DNA-bound protein must occur frequently in the cell but little is known about its outcome. Here, we developed a multi-color single molecule fluorescence approach to simultaneously monitor single stranded (ss) DNA translocation by a helicase and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA binding properties, the two cognate ssDNA binding proteins, RPA1 and RPA2, differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.

Regulation of translocation polarity by helicase domain 1 in SF2B helicasesBiochemical and reverse footprinting studies of the nucleotide excision repair protein XPD show that opposing translocation polarity in superfamily II A and B helicases is an intrinsic property of their respective motor domains, rather than related to different relative DNA binding orientations.

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Many quantitative approaches for analysis of helicase-nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron-sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS containing helicases both in ensemble-based assays and at the single-molecule level. Synthetic oligonucleotides site-specifically labeled with either Cy3 or Cy5 can be used to create a variety of DNA substrates that can be used to characterized DNA binding, as well as helicase translocation and unwinding. Equilibrium binding affinities for ssDNA, duplex and branched DNA substrates can be determined using bulk assays. Identification of preferred cognate substrates, and the orientation and position of the helicase when bound to DNA can also be determined by taking advantage of the intrinsic quencher in the helicase. At the single-molecule level, real-time observation of the helicase translocating along DNA either towards the dye or away from the dye can be used to determine the rate of translocation by the helicase on ssDNA and its orientation when bound to DNA. The use of duplex substrates can reveal the rate of unwinding and processivity of the helicase. Finally, the FeS cluster can be used to visualize protein-protein interactions, and to examine the interplay between helicases and other DNA binding proteins on the same DNA substrate.

DNA helicases are members of a larger class of enzymes composed of molecular motors that couple the energy of NTP binding and hydrolysis to directional translocation along a nucleic acid lattice. Structurally and mechanistically related helicases perform drastically different roles in DNA metabolism by associating with various macromolecular machineries that orchestrate DNA processing events. Within these macromolecular ensembles, DNA helicase translocation is coupled to separation of duplex DNA into two single strands, which is necessary for DNA replication, repair, recombination and transcription. Translocation by DNA helicases can also result in the disassembly of protein–nucleic acid complexes formed during DNA metabolism. One major consequence of cellular dependence on DNA helicases is that defects in these enzymes result in a broad spectrum of disorders usually characterized by premature aging, susceptibility to cancer, and other diseases normally associated with aging.

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