Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

Qi, Zhi; Pugh, Robert A.; Spies, Maria; Chemla, Yann R.

doi:10.7554/elife.00334

Cited by 76 publications

(152 citation statements)

References 74 publications

(152 reference statements)

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“…S1). This behavior may reflect sequence-dependent unwinding and/or pause kinetics, as has been observed for other superfamily (SF) 1 and 2 helicases including hepatitis virus NS3 helicase (18), XPD helicase (19), and RecBCD helicase (20). Additional experiments will be required to characterize the sequence dependence of RecQ unwinding and pausing, and the role of the HRDC domain in these processes.…”

Section: Resultsmentioning

confidence: 84%

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Harami

Seol

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.RecQ | helicase | magnetic tweezers | single molecule | DNA unwinding

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Section: Resultsmentioning

confidence: 84%

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Harami

Seol

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast to E1, the T7 helicase can unwind hundreds of base pairs at a uniform rate before slippage occurs, whereas a majority of E1 unwinding events are interrupted by slippage even for relatively short dsDNA of 34 bp. There are other cases in which repetitive translocation was observed (37)(38)(39)(40)(41)(42)(43)(44)(45). However, these were due to the helicase reaching the end of the track and snapping back.…”

Section: Discussionmentioning

confidence: 99%

Dynamic look at DNA unwinding by a replicative helicase

Lee

Syed

Enemark

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.ATPase | molecular motors D NA replication is the most fundamental of all of life's processes. One of the key requisites in the initiation of replication is the separation of the two strands of the double helix, which is carried out by a hexameric helicase. Despite their prominent roles in biology, some of the basic aspects of these helicases, whether they use a strand-exclusion mechanism or whether they translocate along double-stranded DNA, for example, have been subjects of considerable debate (1, 2). Viral replicative helicases, such as SV40 Large-T antigen (LTag) and papillomavirus E1, have provided the opportunity to study some of these basic features largely owing to their homohexameric architecture. These viral helicases recognize their respective origin of DNA replication (ori) through their dsDNA-binding domains (DBDs) and assemble in a stepwise fashion, ultimately forming double-hexameric (DH) structures on their ori and unwind the DNA bidirectionally.E1 consists of an N-terminal domain, a DBD, an oligomerization domain (OD), a helicase/ATPase domain (HD), and a C-terminal acidic tail (Fig. 1A). Biochemical and structural data have demonstrated that the DBDs bound to the pseudopalindromic E1 binding site are at the center of the double hexamer and that the helicase domains that bind to the flanks of the ori are on either end in a head-to-head arrangement (3, 4). This arrangement is supported by EM studies of LTag that show a dumbbell-shaped structure for the DH (5, 6), with each half of the dumbbell containing two lobes: a larger HD outer lobe and a smaller DBD inner lobe. The assembled DH appears to place the HD of E1 proximal to the dsDNA to be unwound.However, in the structure of the E1 helicase with ssDNA and Mg 2+ -ADP, the ssDNA is oriented such that the N-terminal part of the polypeptide (the OD) is closest to the 5′ end of the DNA (7). The 3′→5′ polarity of E1 helicase indicates that the translocating helicase moves with the N-terminal OD leading and the C-terminal helicase domain trailing, or alternatively, that the DNA is pumped through the helicase from the OD side...

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“…The question remained unanswered for most helicases due to the lack of proper methods that can interrogate the stepping kinetics of helicases [5][6][7]. Optical tweezers (OT) are so far the most reliable technique to study single helicases with 0.5-bp resolution [8][9][10]. However, high-resolution measurements with OT require complicated instrumentation that is accessible to few laboratories only.…”

mentioning

confidence: 99%

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

Lin

Da-Guan

et al. 2017

Phys. Rev. Lett.

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Single-molecule FRET is widely used to study helicases by detecting distance changes between a fluorescent donor and an acceptor anchored to overhangs of a forked DNA duplex. However, it has lacked single-base pair (1-bp) resolution required for revealing stepping dynamics in unwinding because FRET signals are usually blurred by thermal fluctuations of the overhangs. We designed a nanotensioner in which a short DNA is bent to exert a force on the overhangs, just as in optical/magnetic tweezers. The strategy improved the resolution of FRET to 0.5 bp, high enough to uncover the differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.Helicases are motor proteins involved in almost every aspect of nucleic acid metabolism [1][2][3][4]. When a helicase is loaded onto one of the overhangs (i.e., the tracking strand) of a forked DNA duplex and unwinds it, two nucleotides would be released per base pair unwound, leading to an increase in end-to-end distance of the overhangs. It is of great interest to know how a helicase uses the discrete energy derived from NTP hydrolysis to unwind DNA. The question remained unanswered for most helicases due to the lack of proper methods that can interrogate the stepping kinetics of helicases [5][6][7]. Optical tweezers (OT) are so far the most reliable technique to study single helicases with 0.5-bp resolution [8][9][10]. However, high-resolution measurements with OT require complicated instrumentation that is accessible to few laboratories only. In addition, OT measures one molecule at a time so that the throughput is usually very low. smFRET is a high-throughput technique for helicase assays. Using wide-field fluorescence microscopy, one can routinely record signals in parallel from hundreds of single molecules tethered to a surface [11][12][13][14]. To date,

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Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

Cited by 76 publications

References 74 publications

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Dynamic look at DNA unwinding by a replicative helicase

Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

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