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• W1996984611 abstract "Helicases use the energy derived from nucleoside triphosphate hydrolysis to unwind double helices in essentially every metabolic pathway involving nucleic acids. Earlier crystal structures have suggested that DNA helicases translocate along a single-stranded DNA in an inchworm fashion. We report here a series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates. These structures reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand. Thus DNA unwinding is achieved by a two-part power stroke in a combined wrench-and-inchworm mechanism. The rotational angle and translational distance of DNA define the unwinding step to be 1 base pair per ATP hydrolyzed. Finally, a gateway for ssDNA translocation and an alternative strand-displacement mode may explain the varying step sizes reported previously. Helicases use the energy derived from nucleoside triphosphate hydrolysis to unwind double helices in essentially every metabolic pathway involving nucleic acids. Earlier crystal structures have suggested that DNA helicases translocate along a single-stranded DNA in an inchworm fashion. We report here a series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates. These structures reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand. Thus DNA unwinding is achieved by a two-part power stroke in a combined wrench-and-inchworm mechanism. The rotational angle and translational distance of DNA define the unwinding step to be 1 base pair per ATP hydrolyzed. Finally, a gateway for ssDNA translocation and an alternative strand-displacement mode may explain the varying step sizes reported previously. The discovery of DNA double helix immediately presented the challenge of separating the intertwined strands for replication. DNA helicases were first isolated and characterized in the 1970s as DNA-dependent ATPases (Abdel-Monem et al., 1977Abdel-Monem M. Chanal M.C. Hoffmann-Berling H. DNA unwinding enzyme II of Escherichia coli. 1. Purification and characterization of the ATPase activity.Eur. J. Biochem. 1977; 79: 33-38Crossref PubMed Scopus (41) Google Scholar, Richet and Kohiyama, 1976Richet E. Kohiyama M. Purification and characterization of a DNA-dependent ATPase from Escherichia coli.J. Biol. Chem. 1976; 251: 808-812Abstract Full Text PDF PubMed Google Scholar, Wickner et al., 1974Wickner S. Wright M. Hurwitz J. Association of DNA-dependent and -independent ribonucleoside triphosphatase activities with dnaB gene product of Escherichia coli.Proc. Natl. Acad. Sci. USA. 1974; 71: 783-787Crossref PubMed Scopus (59) Google Scholar). Since then a large variety of DNA and RNA helicases have been discovered and characterized. They are implicated in processes ranging from replication to translation (Gorbalenya and Koonin, 1993Gorbalenya A.E. Koonin E.V. Helicases: amino acid sequence comparisons and structure—function relationships.Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (990) Google Scholar, Singleton and Wigley, 2002Singleton M.R. Wigley D.B. Modularity and specialization in superfamily 1 and 2 helicases.J. Bacteriol. 2002; 184: 1819-1826Crossref PubMed Scopus (165) Google Scholar, von Hippel and Delagoutte, 2003von Hippel P.H. Delagoutte E. Macromolecular complexes that unwind nucleic acids.Bioessays. 2003; 25: 1168-1177Crossref PubMed Scopus (28) Google Scholar) and more recently in ATP-dependent chromatin remodeling (Becker and Horz, 2002Becker P.B. Horz W. ATP-dependent nucleosome remodeling.Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (605) Google Scholar). In a broad sense, helicases can be viewed as motor proteins that translocate along double- or single-stranded nucleic acids. Based on sequence analysis, helicases have been grouped into six families: three superfamilies (SF1 to 3), two small families (Rho and DnaB-like), and a branch in the AAA+ family (Gorbalenya and Koonin, 1993Gorbalenya A.E. Koonin E.V. Helicases: amino acid sequence comparisons and structure—function relationships.Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (990) Google Scholar, Iyer et al., 2004Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. Evolutionary history and higher order classification of AAA+ ATPases.J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (595) Google Scholar). UvrD, originally known as DNA helicase II in E. coli (Hickson et al., 1983Hickson I.D. Arthur H.M. Bramhill D. Emmerson P.T. The E. coli uvrD gene product is DNA helicase II.Mol. Gen. Genet. 1983; 190: 265-270Crossref PubMed Scopus (49) Google Scholar), is the founding member of SF1 and unwinds DNA in the 3′→ 5′ direction (Matson and George, 1987Matson S.W. George J.W. DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities.J. Biol. Chem. 1987; 262: 2066-2076Abstract Full Text PDF PubMed Google Scholar). UvrD plays a critical role in replication, recombination, and repair of ultraviolet (UV) damage and mismatched base pairs (Arthur and Lloyd, 1980Arthur H.M. Lloyd R.G. Hyper-recombination in uvrD mutants of Escherichia coli K-12.Mol. Gen. Genet. 1980; 180: 185-191Crossref PubMed Scopus (80) Google Scholar, Bruand and Ehrlich, 2000Bruand C. Ehrlich S.D. UvrD-dependent replication of rolling-circle plasmids in Escherichia coli.Mol. Microbiol. 2000; 35: 204-210Crossref PubMed Scopus (70) Google Scholar, Dao and Modrich, 1998Dao V. Modrich P. Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex.J. Biol. Chem. 1998; 273: 9202-9207Crossref PubMed Scopus (111) Google Scholar, Ogawa et al., 1968Ogawa H. Shimada K. Tomizawa J. Studies on radiation-sensitive mutants of E. coli. I. Mutants defective in the repair synthesis.Mol. Gen. Genet. 1968; 101: 227-244Crossref PubMed Scopus (129) Google Scholar, van de Putte et al., 1965van de Putte P. van Sluis C.A. van Dillewijn J. Rorsch A. The location of genes controlling radiation sensitivity in Escherichia coli.Mutat. Res. 1965; 2: 97-110Crossref PubMed Scopus (49) Google Scholar, Veaute et al., 2005Veaute X. Delmas S. Selva M. Jeusset J. Le Cam E. Matic I. Fabre F. Petit M.A. UvrD helicase, unlike Rep helicase, dismantles RecA nucleoprotein filaments in Escherichia coli.EMBO J. 2005; 24: 180-189Crossref PubMed Scopus (203) Google Scholar). SF1 and SF2 members are distinct from other helicases by sharing seven conserved sequence motifs (Gorbalenya and Koonin, 1993Gorbalenya A.E. Koonin E.V. Helicases: amino acid sequence comparisons and structure—function relationships.Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (990) Google Scholar, Hodgman, 1988Hodgman T.C. A new superfamily of replicative proteins.Nature. 1988; 333: 22-23Crossref PubMed Scopus (320) Google Scholar). These motifs are involved in nucleoside triphosphate (NTP) binding and are located at the interface between two RecA-like domains in the structures of PcrA, Rep, and RecBCD of SF1 and RecG, RecQ, UvrB, eIF4A, Swi2/SNF2, and viral NS3 helicases of SF2 (Bernstein et al., 2003Bernstein D.A. Zittel M.C. Keck J.L. High-resolution structure of the E.coli RecQ helicase catalytic core.EMBO J. 2003; 22: 4910-4921Crossref PubMed Scopus (209) Google Scholar, Durr et al., 2005Durr H. Korner C. Muller M. Hickmann V. Hopfner K.P. X-ray structures of the Sulfolobus solfataricus SWI2/SNF2 ATPase core and its complex with DNA.Cell. 2005; 121: 363-373Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, Singleton et al., 2004Singleton M.R. Dillingham M.S. Gaudier M. Kowalczykowski S.C. Wigley D.B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.Nature. 2004; 432: 187-193Crossref PubMed Scopus (305) Google Scholar, Singleton and Wigley, 2002Singleton M.R. Wigley D.B. Modularity and specialization in superfamily 1 and 2 helicases.J. Bacteriol. 2002; 184: 1819-1826Crossref PubMed Scopus (165) Google Scholar). The crystal structures of a UvrD homolog, PcrA, complexed with DNA-SO42− or DNA-AMPPNP (an ATP analog) (Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar) showed that (1) single-stranded DNA (ssDNA) binds across the surface of the two RecA-like domains (1A and 2A), (2) 1A and 2A rotate toward each other when bound to ATP and open up after ATP hydrolysis, and (3) with the domain movement PcrA translocates along the ssDNA like an inchworm with alternating tight and loose interactions at two contact point. Structures of the Rep and NS3 helicases reveal a similar arrangement of the ATP-binding domains and single-stranded nucleic acid (Kim et al., 1998Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding.Structure. 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, Korolev et al., 1997Korolev S. Hsieh J. Gauss G.H. Lohman T.M. Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.Cell. 1997; 90: 635-647Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Kinetic studies of PcrA (Dillingham et al., 2000Dillingham M.S. Wigley D.B. Webb M.R. Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed.Biochemistry. 2000; 39: 205-212Crossref PubMed Scopus (190) Google Scholar) support the structural finding that the step size of ssDNA translocation is one nucleotide advanced per ATP hydrolyzed. Despite extensive biochemical and structural characterization of SF1 and other helicases, the mechanism for unwinding a DNA or RNA duplex remains highly controversial. To unwind a double helix, contacts between the helicase and the duplex region of DNA and a rotational movement are likely required in addition to ssDNA translocation, but no DNA rotation has yet been detected. PcrA and Rep helicases contain domains 1B and 2B in addition to 1A and 2A. Domain 2B of PcrA, which undergoes an ∼150° rotation upon binding to a double- and single-stranded (ds-ss) DNA junction and transforms the helicase from an “open” to “closed” state (Movie S1), has been shown to be essential for duplex binding and unwinding in solution (Soultanas et al., 2000Soultanas P. Dillingham M.S. Wiley P. Webb M.R. Wigley D.B. Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.EMBO J. 2000; 19: 3799-3810Crossref PubMed Scopus (126) Google Scholar). In the PcrA-DNA-AMPPNP cocrystal structure, the dsDNA is contacted by domains 1B and 2B, but the base pairs adjacent to the ds-ss junction are disrupted, and in the PcrA-DNA-SO42− structure this portion of the dsDNA is disordered (Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). The protein-dsDNA contact was therefore proposed to melt duplex and facilitate ssDNA translocation (Soultanas et al., 2000Soultanas P. Dillingham M.S. Wiley P. Webb M.R. Wigley D.B. Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism.EMBO J. 2000; 19: 3799-3810Crossref PubMed Scopus (126) Google Scholar). However, when domain 2B is deleted in the homologous Rep helicase, the mutant Rep retains helicase activity both in vitro and in vivo (Cheng et al., 2002Cheng W. Brendza K.M. Gauss G.H. Korolev S. Waksman G. Lohman T.M. The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity.Proc. Natl. Acad. Sci. USA. 2002; 99: 16006-16011Crossref PubMed Scopus (54) Google Scholar). Structurally, the 2B domain can assume open or closed conformations when Rep is bound to a ssDNA (Korolev et al., 1997Korolev S. Hsieh J. Gauss G.H. Lohman T.M. Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.Cell. 1997; 90: 635-647Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). These differences highlight not only the puzzling role of the 2B domain but also the absence of a complete model of how DNA duplex is unwound. Furthermore, although PcrA is functional as a monomer, single-turnover kinetic studies of UvrD suggest that a dimeric form is required for DNA unwinding (Maluf et al., 2003Maluf N.K. Fischer C.J. Lohman T.M. A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro.J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (156) Google Scholar). Finally, with different types of kinetic assay and data analysis, different values of step size (defined as the number of base pairs unwound and translocated per ATP hydrolyzed) have emerged, varying from 1 to 6 bp for a given helicase (Ali and Lohman, 1997Ali J.A. Lohman T.M. Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase.Science. 1997; 275: 377-380Crossref PubMed Scopus (224) Google Scholar, Lohman et al., 2003Lohman T.M. Hsieh J. Maluf N.K. Cheng W. Lucius A.L. Fischer C.J. Brendza K.M. Korolev S. Waksman G. DNA helicases, motors that move along nucleic acids: lessons from the SF1 helicase superfamily.in: Hackney D.D. Tamanoi F. Energy coupling and molecular motors (The Enzymes). Elsevier Academic Press, San Diego2003: 304-364Google Scholar, Lucius and Lohman, 2004Lucius A.L. Lohman T.M. Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E.coli RecBCD helicase-catalyzed DNA unwinding.J. Mol. Biol. 2004; 339: 751-771Crossref PubMed Scopus (41) Google Scholar, Roman and Kowalczykowski, 1989Roman L.J. Kowalczykowski S.C. Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA.Biochemistry. 1989; 28: 2873-2881Crossref PubMed Scopus (64) Google Scholar). Even with the simplified ssDNA translocation analyses of monomeric UvrD, step sizes of 2 and 4 were reported by a single group (Fischer et al., 2004Fischer C.J. Maluf N.K. Lohman T.M. Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA.J. Mol. Biol. 2004; 344: 1287-1309Crossref PubMed Scopus (162) Google Scholar, Lohman et al., 2003Lohman T.M. Hsieh J. Maluf N.K. Cheng W. Lucius A.L. Fischer C.J. Brendza K.M. Korolev S. Waksman G. DNA helicases, motors that move along nucleic acids: lessons from the SF1 helicase superfamily.in: Hackney D.D. Tamanoi F. Energy coupling and molecular motors (The Enzymes). Elsevier Academic Press, San Diego2003: 304-364Google Scholar). We have determined at atomic resolution ten crystal structures of UvrD-DNA complexes, which represent three distinct ATP hydrolysis states: (1) binary complexes with or without a bound SO42−, (2) ternary complexes with a nonhydrolyzable ATP analog (AMPPNP), and (3) ternary complexes with an ATP hydrolysis intermediate (ADP·MgF3). Each ds-ss DNA junction is bound by one UvrD monomer, and each structural state presents different interactions between UvrD and the single- and double-stranded regions of DNA. Together they reveal a previously unknown unidirectional rotation and translation of dsDNA and a gateway for ssDNA translocation. The structures and accompanying functional studies lead to the proposal of a combined wrench-and-inchworm mechanism for DNA unwinding by UvrD at the step size of 1 base pair. E. coli UvrD was crystallized with multiple self-complementary oligonucleotides that form 18 to 28 base pair (bp) duplexes flanked by 3′, 7 nucleotide (nt) overhangs and, in some instances, 5′, 1 nt overhangs (Figure S1). In each case, the crystallographic asymmetric units were composed of one such DNA and two bound UvrD monomers, one at each ds-ss junction. Crystals of UvrD-DNA binary complexes (28 bp + 7 nt), UvrD-DNA-AMPPNP (1 nt + 18 bp + 7 nt), and the UvrD-DNA-ADP·MgF3 ternary complexes (18 bp + 7 nt) diffracted X-rays to 3.0, 2.6, and 2.2 Å, respectively, the highest resolution structures determined thus far for helicase-DNA complexes (Figure 1 and Table 1). Residues 1–646 of UvrD, the entire duplex region of DNA, and the first 5 or 6 nt from the ds-ss junction are modeled in each structure. Residues 647–662 of UvrD form random coils but are often traceable in the ternary complex structures. In all UvrD-DNA cocrystal structures, the two UvrD molecules in an asymmetric unit are essentially identical, with rmsd's of all Cα atoms varying between 0.3 and 0.5 Å. In addition, each reaction state was observed in multiple crystal lattices to verify that a particular conformation is independent of crystal packing (Figure S1). The differences between the binary and two ternary complexes are substantial (Figure 1).Table 1Data Collection and Refinement StatisticsSulfate-Bound FormNt-FreeAMPPNPADP·MgF3Data CollectionSpace groupP21212P212121P21P21Resolution (Å)aValues for the highest resolution shell are indicated in parentheses.50–2.9 (3.00–2.90)50–3.0 (3.11–3.00)50–2.6 (2.69–2.6)50–2.2 (2.28–2.2)Completeness (%)aValues for the highest resolution shell are indicated in parentheses.99.2 (92.5)87.0 (57.9)89.7 (51.5)93.5 (63.0)RmergeaValues for the highest resolution shell are indicated in parentheses., bRmerge = ∑h∑i|I(h)i − <I(h)>|/ ∑h∑iI(h)i, where I(h) is the intensity of reflection h, ∑h is the sum over all reflections, and ∑i is the sum over i measurements of reflection h.8.8 (58.4)7.7 (46.9)6.1 (20.6)6.8 (29.3)I / σ(I)aValues for the highest resolution shell are indicated in parentheses.21.7 (2.17)22.8 (2.56)23.3 (4.1)30.1 (2.67)RefinementUnique reflections48,03737,49461,012102,202Protein + DNA atoms10,81511,48410,96911,214Metal + Solvent atoms8723182577R-factor (Rfree) (%)aValues for the highest resolution shell are indicated in parentheses., cR factor = ∑||Fo| − |Fc||/∑|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes. Rfree is calculated for a randomly chosen 10% of reflections that were not used for structure refinement and R factor is calculated for the remaining reflections.23.7 (29.6)23.0 (28.5)21.5 (25.7)21.0 (24.0)Average B-factor (Wilson) (Å2)55.31 (32.36)76.27 (61.35)77.04 (46.42)56.05 (42.29)Rms deviations Bonds (Å)0.00800.00800.00670.0060 Angles (°)1.381.411.231.13a Values for the highest resolution shell are indicated in parentheses.b Rmerge = ∑h∑i|I(h)i − <I(h)>|/ ∑h∑iI(h)i, where I(h) is the intensity of reflection h, ∑h is the sum over all reflections, and ∑i is the sum over i measurements of reflection h.c R factor = ∑||Fo| − |Fc||/∑|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes. Rfree is calculated for a randomly chosen 10% of reflections that were not used for structure refinement and R factor is calculated for the remaining reflections. Open table in a new tab UvrD shares 42% sequence identity with PcrA and 37% with Rep. Like these two SF1 helicases, UvrD contains four structural domains 1A (1–89, 215–280 aa), 1B (90–214 aa), 2A (281–377, 551–647 aa), and 2B (378–550 aa) and adopts the closed conformation observed for the PcrA-DNA complexes (Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). Domains 1A and 2A form the core of the helicase responsible for ATP binding and hydrolysis (Figure 1). The ds-ss DNA junction associated with each UvrD molecule assumes the “L” shape, and the duplex and single-stranded portions are roughly orthogonal to each other. The 3′-ssDNA tail is bound across domains 1A and 2A at their interface with 1B and 2B. Domains 1B and 2B interact with the DNA duplex along one side, covering 14 bp in the ternary complexes or 16 bp in the binary complexes (Figure 1). When the duplex is only 18 bp in length as in the AMPPNP ternary complexes, the central 10 bp are sandwiched between two noncontacting UvrD molecules (Figure S1A). Surprisingly, all DNA duplexes bound to UvrD are fully base paired and in regular B form with no sign of melting or distortion. The seven sequence motifs (I, Ia, II–VI) (Gorbalenya and Koonin, 1993Gorbalenya A.E. Koonin E.V. Helicases: amino acid sequence comparisons and structure—function relationships.Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (990) Google Scholar) and the recently identified Q motif (Tanner et al., 2003Tanner N.K. Cordin O. Banroques J. Doere M. Linder P. The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis.Mol. Cell. 2003; 11: 127-138Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar) conserved among SF1 and SF2 are involved in ATP binding as reported (Theis et al., 1999Theis K. Chen P.J. Skorvaga M. Van Houten B. Kisker C. Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.EMBO J. 1999; 18: 6899-6907Crossref PubMed Scopus (159) Google Scholar, Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar) (Figure 2). Motifs Ia, III, and V are also involved in ssDNA binding. In addition to these eight motifs and motif IVa reported to be unique in SF1 (Korolev et al., 1998Korolev S. Yao N. Lohman T.M. Weber P.C. Waksman G. Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases.Protein Sci. 1998; 7: 605-610Crossref PubMed Scopus (100) Google Scholar), we have identified seven new sequence motifs conserved among UvrD homologs including yeast Srs2. They are Ib, Ic, Id, IVb, IVc, Va, and VIa (Figure 2A). These conserved residues participate in DNA binding or domain 1B and 2B interactions (Figure 2B). The unprecedented high resolution of the UvrD structures allows examination of the atomic details of ATP binding- and hydrolysis-induced protein and DNA movement. The ATP analog, AMPPNP, is bound in the cleft between domains 1A and 2A (Figure 2). The adenine base is sandwiched between Y283 (motif IV) and R37 (motif I) and is specifically selected by Q14 (Q motif) through bifurcated hydrogen bonds. GTP does not support helicase activity (Figure S2) (Matson and Kaiser-Rogers, 1990Matson S.W. Kaiser-Rogers K.A. DNA helicases.Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (330) Google Scholar). The 3′ OH of the ribose is hydrogen bonded to E566 (motif V), but the 2′ OH is only weakly hydrogen bonded to R37, which explains why UvrD can use both ATP and dATP (Figure S2) (Matson and Kaiser-Rogers, 1990Matson S.W. Kaiser-Rogers K.A. DNA helicases.Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (330) Google Scholar). Four basic residues, K35 (motif I or Walker A), R73 (Ia), R284 (IV), and R605 (VI), coordinate the triphosphate moiety, in particular the γ phosphate (Figure 2C). A single Mg2+ ion essential for ATP hydrolysis is coordinated directly by the β and γ phosphates and T36 (I), and by D220 and E221 (motif II or Walker B) through water. E221 is also well positioned to serve as a general base to deprotonate the water molecule that is oriented by Q251 (III) for the in-line nucleophilic attack (Figure 2C). Substitutions of the equivalent of Q251 in PcrA resulted in reduced ATPase activity (Dillingham et al., 1999Dillingham M.S. Soultanas P. Wigley D.B. Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation.Nucleic Acids Res. 1999; 27: 3310-3317Crossref PubMed Scopus (82) Google Scholar). In the attempt to crystallize UvrD-DNA-ADP complexes, we fortuitously made ADP·MgF3 complexes after NaF was added to improve the crystal growth. NaF reacted with MgCl2 and ADP in the crystallization solution and formed ADP·MgF3, which produced the best diffracting crystals of UvrD-DNA complexes (Table 1; Figure S1). MgF3 mimics the planar structure of the pentacovalent phosphate in the transition state, and the distances between the Mg (P mimic) and the apical oxygen atoms (attacking and leaving groups) are 1.9–2.0 Å (Figure 2D). MgF3 is believed to form a more authentic transition state analog than AlF(x) or BeF(x), of which x is either 3 or 4 (Graham et al., 2002Graham D.L. Lowe P.N. Grime G.W. Marsh M. Rittinger K. Smerdon S.J. Gamblin S.J. Eccleston J.F. MgF(3)(-) as a transition state analog of phosphoryl transfer.Chem. Biol. 2002; 9: 375-381Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The arrangement of UvrD and DNA in the AMPPNP versus the ADP·MgF3 bound state is almost identical except for the linkers between domains 2A and 2B and the 3′ end of ssDNA (see details later). AMPPNP binding induces an ∼20° rotation between domain 2A and the remaining three domains (1A, 1B, and 2B), and the distance between the respective centers of mass is reduced by 3.3 Å (Figure 3). Domains 1A and 1B are linked by a shared hydrophobic core, and domains 1B and 2B by salt bridges (K389 to D115 and R396 to D118). These three domains of UvrD move as one unit (Figure S3). In contrast, the movement of domain 2B in PcrA is uncoupled from that of domains 1A and 1B (Velankar et al., 1999Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). The fully ordered dsDNA in the UvrD structures likely stabilizes the 2B domain and 2B-1B interactions (Figure 1). Local structural changes between the binary and ternary complexes also occur in the ATP-binding loops and the loops that contact DNA. After ATP hydrolysis, the domain rotation is presumably reversed during release of ADP and Pi. The domain rotation axis passes through the Cα of W256 (motif III, located at the 1A and 2A interface) and is 15 Å from the helical axis of dsDNA at an ∼25° angle (Figure 3). The domain movement in UvrD is coupled with the directional movement of the DNA duplex. Three helix-loop-helix (HLH) structures emanating from domain 2B (α2-α3, α4-α5, and α7-α8) alternately contact each DNA strand in the duplex, and the N terminus of the second helix of each HLH is in close contact with the DNA backbone (Figure 4A). The first HLH (2B-α2-α3), which contains the PxxGIGxxT sequence (motif IVc or GIG), is conserved among UvrD homologs (Figure 2A). The backbone amide groups of the two Gly's form tight hydrogen bonds with the DNA backbone (Figure 4B). Interestingly, the first Gly of GIG is replaced by Glu in Rep (Figure 2A), and Rep binds duplex DNA poorly (Myong et al., 2005Myong S. Rasnik I. Joo C. Lohman T.M. Ha T. Repetitive shuttling of a motor protein on DNA.Nature. 2005; 437: 1321-1325Crossref PubMed Scopus (213) Google Scholar). Near the ds-ss junction, a fourth HLH from domain 1B (α2-α3) is in van der Waals contact with both DNA strands in the minor groove (Figure 4A). Upon AMPPNP binding, the duplex moves with domains 1A/1B/2B toward 2A. The movement includes a 3.3 Å translation and an ∼20° left-handed rotation that untwists the double helix (Figure 1, Figure 3). At the ds-ss junction, a β hairpin (614–626 aa, motif VIa) in the 2A domain, which we call the separation pin, buttresses the end of the duplex. Y621 on its tip forms a π ring stack with the first base pair (−1) of the DNA duplex in the binary complex (Figure 4C). When pressed against the separation pin in the AMPPNP or ADP·MgF3 ternary complexes, the −1 base pair becomes unpaired (Movie S2), and the side chain of Y621 rotates to a vertical position as if to facilitate the newly unpaired base to flip out (Figure 4D). As the −1 bp is unpaired, the number of base pairs between the GIG motif and separation pin is reduced from 10 to 9 in the ternary complexes (Figure 1, Figure 4). Upon binding of AMPPNP, the first three nucleotides of the existing 3′ ssDNA (numbered +1 onward from the ds-ss junction) retain the same positions as in the binary complexes, so the newly unpaired base (−1) on this strand bulges out (Figure 1, Figure 5). The −1 nt on the partner strand is disordered. The +1 to +3 nt interact extensively with domains 2A (M584, R355, H560) and 1A (Y254 and W256 of motif III) (Figure 5A). Since W256 intersects the domain rotation axis, it remains immobile throughout the ATPase cycle. W256 stacks with the bases of the +1 and +2 nt and buttresses the +3 base (Figure 5A). Motif III, which is essential for the helicase activity (Dillingham et al., 1999Dillingham M.S. Soultanas P. Wigley D.B. Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation.Nucleic Acids Res. 1999; 27: 3310-3317Crossref PubMed Scopus (82) Google Scholar), functions as an “anchor” for ssDNA during" @default.
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• W1996984611 date "2006-12-01" @default.
• W1996984611 modified "2023-10-17" @default.
• W1996984611 title "UvrD Helicase Unwinds DNA One Base Pair at a Time by a Two-Part Power Stroke" @default.
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• W1996984611 doi "https://doi.org/10.1016/j.cell.2006.10.049" @default.
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