Mechanisms of Helicases

Abstract

Helicase protein was first discovered in Escherichia coli in 1976 (1Abdel-Monem M. Durwald H. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 441-449Crossref PubMed Scopus (138) Google Scholar, 2Abdel-Monem M. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 431-440Crossref PubMed Scopus (107) Google Scholar). Since then RNA and DNA helicases with diverse functions have been found in all organisms. Helicases couple the chemical energy of NTP 2The abbreviations used are: NTP, nucleoside triphosphate; SF, superfamily; HCV, hepatitis C virus; L-Tag, large T-antigen. 2The abbreviations used are: NTP, nucleoside triphosphate; SF, superfamily; HCV, hepatitis C virus; L-Tag, large T-antigen. binding and hydrolysis to separate the complementary strands of double-stranded nucleic acids, remove nucleic acid-associated proteins, or catalyze homologous DNA recombination. Although helicases display these activities in isolation, most work efficiently as part of a larger protein complex. Interests in understanding helicase mechanisms stem not only from their importance in various cellular processes. Helicases also serve as a model system to understand NTPase-coupled motors. The helicase function is required for efficient and accurate replication, repair, and recombination of the genome. Similarly, helicase functions facilitate RNA metabolic processes such as transcription, ribosome biogenesis, translation, RNA splicing, RNA editing, RNA transport, and RNA degradation. The structure, function, and mechanisms of helicases have been widely discussed in the literature (3Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (335) Google Scholar, 4Lohman T.M. J. Biol. Chem. 1993; 268: 2269-2272Abstract Full Text PDF PubMed Google Scholar, 5Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (670) Google Scholar, 6West S.C. 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Ganesan S. Kass E.M. Drapkin R. Grossman S. Wahrer D.C. Sgroi D.C. Lane W.S. Haber D.A. Livingston D.M. Cell. 2001; 105: 149-160Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Many of the implicated helicases such as the RecQ family of helicases are involved in DNA repair and in processes assuring genome stability. To link the defect in a particular helicase to the human disease will require defining the cellular roles of these helicases and determining the exact steps in which they are involved in the metabolic pathway. Many viruses code for their own helicases that are essential for viral genome replication (26Kadaré G. Haenni A.L. J. Virol. 1997; 71: 2583-2590Crossref PubMed Google Scholar). Identifying and targeting the unique features of these viral helicases remains a feasible strategy for antiviral therapy (27Kwong A.D. Rao B.G. Jeang K.T. Nat. Rev. Drug Discov. 2005; 4: 845-853Crossref PubMed Scopus (160) Google Scholar, 28Hickman A.B. Dyda F. Curr. Opin. Struct. Biol. 2005; 15: 77-85Crossref PubMed Scopus (81) Google Scholar, 29Crute J.J. Grygon C.A. Hargrave K.D. Simoneau B. Faucher A.M. Bolger G. Kibler P. Liuzzi M. Cordingley M.G. Nat. Med. 2002; 8: 386-391Crossref PubMed Scopus (203) Google Scholar, 30Borowski P. Lang M. Haag A. Schmitz H. Choe J. Chen H.M. Hosmane R.S. Antimicrob. Agents Chemother. 2002; 46: 1231-1239Crossref PubMed Scopus (60) Google Scholar). Based on conserved amino acid sequence motifs, helicases are classified into families and superfamilies (31Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1026) Google Scholar). Helicases of different families share similarities in their three-dimensional folds (RecA-like fold) (32Subramanya H.S. Bird L.E. Brannigan J.A. Wigley D.B. Nature. 1996; 384: 379-383Crossref PubMed Scopus (381) Google Scholar, 33Bird L.E. Subramanya H.S. Wigley D.B. Curr. Opin. Struct. Biol. 1998; 8: 14-18Crossref PubMed Scopus (147) Google Scholar, 34Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). SF1 and SF2 helicases are closely related and typically contain several domains and a single NTP binding site at the interface of two RecA-like domains. Ring-shaped SF3 and SF4 helicases contain an NTP binding site at the interface of adjacent subunits (34Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The nucleic acid binding site in helicases is distinct but allosterically linked to the NTPase site. Examination of sequence, structure, and biochemical properties reveals that helicases of the same superfamily often show different substrate specificity (RNA versus DNA) and directionality of translocation (3′–5′ or 5′–3′). This suggests that small changes in the primary structure of the helicase can be responsible for different substrate specificity and opposite directionality. The structural basis for the substrate specificity and directionality of translocation remains poorly understood. Even though helicases share a similar three-dimensional fold, they assemble into various oligomeric states that are often required to display full activity. The most established form of assembly is exemplified by the hexameric class of helicases (Fig. 1a) in which six subunits assemble to form a ring-shaped structure (T7 gp4, E. coli DnaB, RepA, Rho, MCM, SV40-LTag) (8Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (460) Google Scholar). Monomers of ring-shaped helicases (T7 gp4 helicase) are not active in catalyzing NTPase or unwinding reaction, and hexamer formation is essential (35Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The ring-shaped structure is stabilized by the binding of NTP, a metal ion, or both, and by the nucleic acid substrate (8Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (460) Google Scholar). Most studies of ring-shaped helicases are consistent with the nucleic acid bound within the central channel. The enclosure of the nucleic acid by the protein subunits decreases the probability of helicase falling off, thus increasing the ability of the helicase to stay on track. Another advantage of this arrangement is the coupling of NTPase cycles between the hexameric subunits that can increase the efficiency of the NTPase cycles in promoting translocation. Oligomerization is an important strategy for non-ring-shaped SF1 and SF2 helicases as well (Fig. 1, b–e). Even though many helicases function as monomers (Fig. 1b, T4 Dda, HCV NS3h, E. coli RecQ) (36Xu H.Q. Deprez E. Zhang A.H. Tauc P. Ladjimi M.M. Brochon J.C. Auclair C. Xi X.G. J. Biol. Chem. 2003; 278: 34925-34933Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Nanduri B. Byrd A.K. Eoff R.L. Tackett A.J. Raney K.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14722-14727Crossref PubMed Scopus (81) Google Scholar, 38Levin M.K. Wang Y.H. Patel S.S. J. Biol. Chem. 2004; 279: 26005-26012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), most non-ring-shaped helicases studied either require or their activity is greatly enhanced by the formation of dimers or higher oligomers (38Levin M.K. Wang Y.H. Patel S.S. J. Biol. Chem. 2004; 279: 26005-26012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 39Maluf N.K. Fischer C.J. Lohman T.M. J. Mol. Biol. 2003; 325: 913-935Crossref PubMed Scopus (163) Google Scholar, 40Cheng W. Hsieh J. Brendza K.M. Lohman T.M. J. Mol. 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RecBCD helicase consists of two interacting helicases, RecB and RecD, that together are more active than individual helicases (44Dillingham M.S. Spies M. Kowalczykowski S.C. Nature. 2003; 423: 893-897Crossref PubMed Scopus (173) Google Scholar, 45Taylor A.F. Smith G.R. Nature. 2003; 423: 889-893Crossref PubMed Scopus (171) Google Scholar). Many helicases show functional cooperativity and enhanced processivity when multiple molecules of helicases are loaded on the tracking strand (Fig. 1e). These helicases (T4 Dda and HCV NS3h) are functional as monomers, do not form stable oligomers, and do not show cooperativity in NTPase or nucleic acid binding. Yet their activity is enhanced when multiple helicases are loaded on the tracking strand, which is attributed either to prevention of backward helicase slips or simply the availability of additional helicase molecules when one falls off the track (38Levin M.K. Wang Y.H. Patel S.S. J. Biol. Chem. 2004; 279: 26005-26012Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 41Byrd A.K. Raney K.D. Biochemistry. 2005; 44: 12990-12997Crossref PubMed Scopus (50) Google Scholar, 42Tackett A.J. Chen Y. Cameron C.E. Raney K.D. J. Biol. Chem. 2005; 280: 10797-10806Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 46Ali J.A. Maluf N.K. Lohman T.M. J. Mol. Biol. 1999; 293: 815-834Crossref PubMed Scopus (93) Google Scholar). Most helicases need a single-stranded nucleic acid region to bind and to initiate their action of strand separation (Fig. 2, a and b). Once loaded on the strand, they show a directional bias and translocate either 5′–3′ or 3′–5′. Helicases like RecBCD contain two helicases of opposite polarity and can initiate from blunt ended DNA (47Farah J.A. Smith G.R. J. Mol. Biol. 1997; 272: 699-715Crossref PubMed Scopus (49) Google Scholar). Ring-shaped helicases require Y-shaped nucleic acid structures with a loading strand and a non-complementary strand of an optimum length to initiate unwinding (Fig. 2, c–e) (48Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1998; 37: 3116-3136Crossref PubMed Scopus (88) Google Scholar, 49Matson S.W. Tabor S. Richardson C.C. J. Biol. Chem. 1983; 258: 14017-14024Abstract Full Text PDF PubMed Google Scholar, 50Venkatesan M. Silver L.L. Nossal N.G. J. Biol. Chem. 1982; 257: 12426-12434Abstract Full Text PDF PubMed Google Scholar). A bulky adduct can replace the non-complementary strand to facilitate the unwinding process. In the absence of the non-complementary strand or a bulky adduct, ring-shaped helicases (DnaB, T7 gp4, MCM) encircle the duplex DNA and pass over it without unwinding the strands (51Kaplan D.L. J. Mol. Biol. 2000; 301: 285-299Crossref PubMed Scopus (108) Google Scholar, 52Kaplan D.L. Davey M.J. O'Donnell M. J. Biol. Chem. 2003; 278: 49171-49182Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). During unwinding, helicases may assume different nucleic acid binding modes depending on their NTP ligation state (53Wong I. Lohman T.M. Science. 1992; 256: 350-355Crossref PubMed Scopus (166) Google Scholar). Determining the mode of helicase interactions with the nucleic acid substrate during unwinding (Fig. 2) is an important step toward understanding the helicase mechanism. Helicases show different degrees of tolerance to changes in the chemical nature of the loading strand while translocating. Some are sensitive to breaks (discontinuities in sugar phosphate backbone; substituted with ethylene glycol, etc.), to abasic sites, or to electrostatic disruptions (54Eoff R.L. Spurling T.L. Raney K.D. Biochemistry. 2005; 44: 666-674Crossref PubMed Scopus (25) Google Scholar). While unwinding, certain helicases (NPH-II, Dda) show no sensitivity to the nature of the displaced strand (55Tackett A.J. Morris P.D. Dennis R. Goodwin T.E. Raney K.D. Biochemistry. 2001; 40: 543-548Crossref PubMed Scopus (30) Google Scholar, 56Beran R.K. Bruno M.M. Bowers H.A. Jankowsky E. Pyle A.M. J. Mol. Biol. 2006; 358: 974Crossref PubMed Scopus (45) Google Scholar, 57Kawaoka J. Jankowsky E. Pyle A.M. Nat. Struct. Mol. Biol. 2004; 11: 526-530Crossref PubMed Scopus (65) Google Scholar). On the other hand, the nature of the displaced strand appears to influence the activity of HCV NS3 helicase (58Tackett A.J. Wei L. Cameron C.E. Raney K.D. Nucleic Acids Res. 2001; 29: 565-572Crossref PubMed Scopus (62) Google Scholar). During genome replication, repair, or recombination, helicases need to unwind nucleic acids much longer than their binding sites. In such a case, helicase-catalyzed unwinding occurs in a stepwise manner, where the helicase stays on track and catalyzes repeated cycles of base pair separation steps coupled to unidirectional translocation. Many helicases have the ability to translocate unidirectionally along nucleic acids uncoupled from base pair separation. Distinction is therefore made here between translocation mechanisms and base pair separation mechanisms in discussing the proposed models of helicases. Many different mechanisms have been proposed for translocation and base pair separation. All of the mechanisms involve NTPase-coupled nucleic acid affinity changes and a conformational change (power stroke or ratchet) to explain biased movement that results in base pair separation or translocation. The differences in the proposed mechanisms reflect the diverse biochemical properties including the oligomeric state of the helicase, its mode of binding the nucleic acid at the unwinding junction, and the effect of the NTP ligation state on nucleic acid binding properties. Stepping Mechanisms—The stepping mechanisms invoke two nucleic acid binding sites that independently bind and release nucleic acid in response to the signals received from the NTPase site (53Wong I. Lohman T.M. Science. 1992; 256: 350-355Crossref PubMed Scopus (166) Google Scholar, 59Yarranton G.T. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1658-1662Crossref PubMed Scopus (149) Google Scholar, 60Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). In the stepping models, the helicase is always bound to the nucleic acid via one nucleic acid binding site. In an inchworm type stepping model for a monomeric helicase, a cycle of nucleic acid binding, release, and translocation events begins with one helicase site bound tightly to the nucleic acid and the second helicase site bound weakly to the nucleic acid (Fig. 3A). The weak site dissociates from the nucleic acid and in a power stroke motion moves away from the tight site to bind at a position ahead. After the weak site has moved and made tight interactions ahead, the original tight site becomes weak, and as it dissociates from the nucleic acid, in a power stroke motion it moves forward to get close in distance to the site ahead. One cycle in an inchworm stepping mechanism is completed in six conformational changes. The inchworm stepping mechanism is applicable to dimeric helicases as well. In a monomeric helicase, the two nucleic acid binding sites would be present on the same polypeptide and under the control of a single NTP binding site. In dimeric helicase, each subunit would contain a nucleic acid binding site that can be controlled by the NTP binding site of that subunit. In dimeric helicases, therefore, coordinated NTPase activity can lead to coordinated binding and release of nucleic acid. An alternative stepping mechanism (rolling model) for a dimeric helicase has been proposed for DNA unwinding (53Wong I. Lohman T.M. Science. 1992; 256: 350-355Crossref PubMed Scopus (166) Google Scholar). In this model, each of the two subunits of the helicase alternate their binding to single-stranded and duplex DNA as they change their NTP ligation states. In contrast to the inchworm model, where the subunits maintain their relative positioning along the DNA, the subunits in the rolling model take turn in being the trailing or the leading subunit. Brownian Motor Mechanism—A two-state Brownian motor model (Fig. 3B) has been proposed as an alternative to the stepping mechanisms (61Levin M.K. Gurjar M. Patel S.S. Nat. Struct. Mol. Biol. 2005; 12: 429-435Crossref PubMed Scopus (120) Google Scholar, 62Betterton M.D. Julicher F. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2005; 71: 011904Crossref PubMed Scopus (103) Google Scholar). This model invokes Brownian motion and power stroke, and it is based on two conformational states of the helicase. Structural and biochemical studies have identified two distinct conformational states of helicases with weak and tight nucleic acid binding modes resulting from the different NTP ligation states. In the tight state, the helicase-nucleic acid energy profile is deep and sawtooth-shaped, and helicase movement along the nucleic acid is not possible. To translocate the helicase needs to loosen its interactions with the nucleic acid, and this happens when the helicase changes its NTP ligation state (NTP, hydrolysis, or product release). In the weak state, the helicase-nucleic acid energy profile is shallow and symmetric, and the helicase can move in either direction (Brownian motion) or completely dissociate from the nucleic acid (accounting for the observed low processivity of helicases). The short lifetime of the weak state (because of a rapid change in the NTP ligation state) keeps the helicase close to the starting position. When the helicase resumes the tight state, it makes a step forward (power stroke). Those molecules that have fluctuated in the forward direction move ahead and those that have fluctuated in the opposite direction return to the original position. Repetition of these steps leads to net forward movement of the helicase along the nucleic acid. Future challenges lie in distinguishing between the various models of helicase translocation. Accurate measurement of the step size can provide insights into a Brownian motor type versus a deterministic stepping type mechanism of helicase translocation. Base pair separation occurs at the junction of single-stranded and duplex regions. Helicases unwind long stretches of duplex nucleic acids by coupling base pair separation to translocation. Translocation along nucleic acid can occur by any of the above mentioned mechanisms. Depending on how the base pairs are separated, the base pair separation mechanisms are classified as active or passive (9von Hippel P.H. Delagoutte E. Cell. 2001; 104: 177-190Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 62Betterton M.D. Julicher F. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2005; 71: 011904Crossref PubMed Scopus (103) Google Scholar, 63Lohman T.M. Mol. Microbiol. 1992; 6: 5-14Crossref PubMed Scopus (133) Google Scholar). In a passive mechanism, the helicase waits for the base pairs to open spontaneously by thermal fluctuations before it moves and binds the newly opened bases. Because the terminal base pair at the junction opens and closes at a very fast rate (64Gueron M. Leroy J.L. Methods Enzymol. 1995; 261: 383-413Crossref PubMed Scopus (278) Google Scholar), this type of a mechanism is attractive for helicases that can move and occupy one base at a time. The probability of several base pairs opening at the same time near the junction is very low. Therefore, if the helicase needs to move and bind more than one base at a time, it would employ some type of an active mechanism to bring about strand separation in an efficient manner. One simple way for helicases to separate the base pairs at the junction is to translocate one strand through its nucleic acid binding site while excluding the complementary strand (Fig. 4A, wedge, wire stripper, or strand exclusion type mechanism). Many ring-shaped (T7 gp4, DnaB, MCM) and non-ring-shaped helicases (NPH II, Dda) have been proposed to unwind nucleic acid by this mechanism (48Jezewska M.J. Rajendran S. Bujalowski W. Biochemistry. 1998; 37: 3116-3136Crossref PubMed Scopus (88) Google Scholar, 51Kaplan D.L. J. 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Helicases may employ additional ways to destabilize the duplex DNA. Helicases may interact directly with the duplex region near the unwinding junction and distort it prior to fully separating the strands by directional translocation (Fig. 4B, helix-destabilizing mechanism) (53Wong I. Lohman T.M. Science. 1992; 256: 350-355Crossref PubMed Scopus (166) Google Scholar, 60Velankar S.S. Soultanas P. Dillingham M.S. Subramanya H.S. Wigley D.B. Cell. 1999; 97: 75-84Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar). Such a mechanism has been proposed for the PcrA helicase based on a crystal structure that showed interactions of the 2B domain with the duplex DNA. The role of the 2B domain in duplex DNA destabilization is not clear, because deletion of the 2B domain in a highly homologous Rep helicase resulted in enhanced unwinding activity (68Cheng W. Brendza K.M. Gauss G.H. Korolev S. Waksman G. Lohman T.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16006-16011Crossref PubMed Scopus (56) Google Scholar). Many ring-shaped helicases have been proposed to unwind DNA by the strand exclusion model (Fig. 4C), which is essentially the wedge mechanism in Fig. 4A. Many ring-shaped helicases also translocate along duplex DNA to catalyze homologous DNA recombination at a Holliday junction. Ring-shaped helicases are also involved in the initiation of DNA replication, and they have the ability to bind specific sequences at the origin. Although the exact mechanism of unwinding DNA at the origin is not well understood, several models (Fig. 4, D–F, Torsional, SV40 L-Tag model, and Ploughshare) have been proposed to explain DNA unwinding with this mode of DNA binding (69Takahashi T.S. Wigley D.B. Walter J.C. Trends Biochem. Sci. 2005; 30: 437-444Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Three parameters define translocation or strand separation: rate, processivity, and step size. Rate is defined as the number of bases translocated per second, bp unwound per second, or steps per second. Processivity is the probability of making a step forward on the nucleic acid and equal to the rate constant of moving forward divided by the sum of falling off and moving forward rate constants. Processivity is reported in units of per base, per bp, or per step size. Step size can be defined in many ways. Physical step size is the number of bp translocated in a single conformational cycle of the enzyme. Chemical step size is the number of bases translocated or bp unwound per NTP hydrolysis cycle. Kinetic step size is the number of bases translocated or bp unwound between two rate-limiting steps and is obtained from fitting the time course of unwinding measured under single-turnover conditions to a specific stepping model (70Ali J.A. Lohman T.M. Science. 1997; 275: 377-380Crossref PubMed Scopus (231) Google Scholar). 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