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• W2000270965 abstract "DNA helicases play essential roles in key biological processes such as the replication, repair, recombination, and transcription of DNA. Their presence and importance in DNA metabolism was first recognized 20 years ago, when Escherichia coli DNA helicase I was shown to unwind duplex DNA into two single strands. We now know that several human diseases (Xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome, and Werner's syndrome), some of which result in cancer, can be caused by defects in DNA helicases. Surprisingly, DNA helicases remain something of an enigma in modern day molecular biology, since their precise cellular functions and mechanisms of action are not fully understood. Sequence comparisons indicate that many helicases contain the presence of seven distinct highly conserved motifs, two of which correspond to the Walker A and B sites involved in nucleoside triphosphate (NTP) binding. As a class of enzyme, all helicases share the common property of being able to use the energy derived from NTP hydrolysis (usually ATP) to break the hydrogen bonds that hold DNA strands together (for reviews, see8Lohman T.M Bjornson K.P Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (650) Google Scholar, 9Matson S.W Bean D.W George J.W BioEssay. 1994; 16: 13-22Crossref PubMed Scopus (262) Google Scholar). Many also use this energy to fuel their processive translocation along DNA, some at rates exceeding 1000 bp/s, while others act by a nonprocessive (distributive) mechanism. In general, DNA unwinding occurs with a unique directionality, usually defined as a 5′–3′ or 3′–5′ polarity relative to the strand of DNA that is bound by the enzyme. Most helicases function as multimers (either dimers or hexamers), and their oligomeric nature reflects the need for at least two DNA-binding sites; for example, a dimeric protein would be capable of a rolling or inchworm mechanism of translocation. Recent observations, however, point to the presence of a subclass of DNA helicases that possess a common hexameric ring structure (Table 1). It now appears that these hexameric proteins are often integral components of larger protein complexes, and their role within the complex is to provide molecular motor function. Thus, it is naive to think of helicases simply as DNA-unwinding enzymes.Table 1Hexameric DNA HelicasesOrganismProteinSubunit Mass (kDa)Helicase PolarityFunctionT4Gene 41 protein535′–3′Replicative DNA helicaseT7Gene 4 proteinaT7 gp4 exists in two forms, the larger of which also has primase activity.63, 565′–3′Replicative DNA helicase, primaseE. coliDnaB525′–3′Replicative DNA helicaseE. coliRuvBbRequires RuvA protein for junction-binding specificity.375′–3′Branch migration motorE. coliRho465′–3′Transcription terminationSV40Large T antigen923′–5′Replication initiationa T7 gp4 exists in two forms, the larger of which also has primase activity.b Requires RuvA protein for junction-binding specificity. Open table in a new tab In a process such as DNA replication, where single strands are used as templates for DNA polymerase, the need for an unwinding protein that can translocate is readily understood. What is surprising, as shown in new studies of the E. coli DnaB helicase, is that the rate of movement of enzymes at the replication fork is coordinated by interactions between the helicase and the polymerase (6Kim S Dallmann H.G McHenry C.S Marians K.J Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). DnaB protein is the primary replicative helicase in E. coli and is responsible for unwinding duplex DNA ahead of the replication fork. It forms a hexamer composed of six identical subunits (each containing an NTP-binding site), and the subunits associate in the form of a ring (Figure 1A). While it is thought that DNA passes through the center of the ring, cross-linking studies indicate that only one of the six subunits lies in direct contact with DNA (2Bujalowski W Jezewska M.J Biochemistry. 1995; 34: 8513-8519Crossref PubMed Scopus (121) Google Scholar). The conformation of the hexamer is altered by ATP and DNA binding, and two distinct allosteric states (exhibiting 3-fold or 6-fold symmetry) have been observed (13Yu X Jezewska M.J Bujalowski W Egelman E.H J. Mol. Biol. 1996; 259: 7-14Crossref PubMed Scopus (130) Google Scholar). The C3 structure appears to be a trimer of dimers, a result that may relate to observations showing that DnaB contains three high-affinity and three low-affinity ATP-binding sites (1Bujalowski W Klonowska M.M Biochemistry. 1993; 32: 5888-5900Crossref PubMed Scopus (101) Google Scholar). Until recently, the mechanism by which replication of the leading and lagging strands could be coordinated remained a puzzle. However, new work from the Marians laboratory suggests that this may be accomplished by tethering the replicative helicase to the polymerase complex (6Kim S Dallmann H.G McHenry C.S Marians K.J Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Using gel filtration and immunoblotting, direct protein–protein contacts between the τ subunit of pol III holoenzyme and DnaB helicase were demonstrated. By bridging the polymerase dimer with the hexameric helicase, τ subunit coordinates movement of the fork, which then proceeds at ∼1000 nt/s (Figure 2). In the absence of τ, the polymerases and the helicase are uncoupled, such that the polymerase simply follows behind DnaB, as it now unwinds DNA more slowly (35 nt/s). While it is thought that τ mediates dimerization of the leading and lagging strand polymerases, it remains to be determined whether τ induces a conformational change in DnaB that leads to the high rate of translocation. Stimulation of DnaB helicase activity by τ could be due to an allosteric transition within the hexamer that alters its affinity for ATP or DNA, or both, since studies from Bujalowski's laboratory indicate that conformational changes to DnaB (induced by binding a nonhydrolyzable analogue of ATP) result in an affinity for DNA that varies by almost 4 orders of magnitude (5Jezewska M.J Bujalowski W J. Biol. Chem. 1996; 271: 4261-4265Crossref PubMed Scopus (66) Google Scholar). A τ-mediated change in affinity for DNA would exert a profound effect on the ability of DnaB to promote fork movement. Although it is clear that DnaB leads fork movement, it is not known whether both strands of DNA (rather than just one) pass through the center of the hexameric ring. Loading requires the assistance of other proteins, such as DnaC (or λP), which recognize origin sequences, but the mechanism of unwinding that takes place within the ring structure is unknown, as is the way in which rings assemble around DNA. It is possible that preformed rings undergo a transient opening (possibly coupled to the hydrolysis of ATP), or that they assemble on DNA from dimeric species. Hopefully, answers to these questions will lead to progress in understanding the mechanism of action of other hexameric ring helicases. Bacteriophage T7 gene 4 encodes two proteins, a 63 kDa protein (gp4A) with helicase/primase activity and a smaller 56 kDa protein (gp4B) that lacks the N-terminal 63 amino acids and primase activity. Both form hexameric ring structures that exhibit a diameter of 130 Å and a central hole of 25 Å–30 Å through which the DNA passes (3Egelman E.H Yu X Wild R Hingorani M.M Patel S.S Proc. Natl. Acad. Sci. USA. 1995; 92: 3869-3873Crossref PubMed Scopus (247) Google Scholar). The resemblance between gp4 and DnaB is quite remarkable (see Figure 1B). Hexamer formation by gp4 is required for DNA binding and is dependent upon the presence of a NTP. T7 gp4 is rather unusual among helicases in its use of dTTP as the preferred energy source. Image reconstruction reveals that each subunit in the T4 gp4B hexamer is bilobed, with one lobe forming a large ring and the other a smaller ring (Figure 3A). This structural asymmetry is likely to provide the basis for the polarity of helicase action (Figure 3B). Electron microscopic analysis of the RuvB protein of E. coli reveals structures that are again strikingly similar to those observed with T7 gp4, despite the fact that gp4 and RuvB share only limited sequence similarity (which occurs within the seven conserved helicase motifs). When bound to duplex DNA in the presence of a nonhydrolyzable analogue of ATP, RuvB protein forms a dodecameric structure in which two hexameric rings (each exhibiting C6 symmetry in which the six subunits are related by a six-fold rotation axis) are oriented in a bipolar manner (Figure 3C). The outer diameter of each ring is ∼120 Å, and there is a 20 Å–25 Å hole through which the DNA passes (11Stasiak A Tsaneva I.R West S.C Benson C.J.B Yu X Egelman E.H Proc. Natl. Acad. Sci. USA. 1994; 91: 7618-7622Crossref PubMed Scopus (141) Google Scholar). Like T7 gp4 protein, the RuvB hexamer (i.e., half the RuvB dodecamer) appears bilobed, and unwinds DNA with a specific polarity. These overall similarities are intriguing and potentially indicate a general structure for hexameric helicases (3Egelman E.H Yu X Wild R Hingorani M.M Patel S.S Proc. Natl. Acad. Sci. USA. 1995; 92: 3869-3873Crossref PubMed Scopus (247) Google Scholar). RuvB is a DNA damage-inducible protein that is involved in the repair and recombination of DNA. It promotes the branch migration of DNA crossovers (also known as Holliday junctions) (Figure 4A). RuvB does not act alone; it interacts with RuvA which is transcribed from the same operon. RuvA provides DNA-binding specificity by targeting RuvB to the Holliday junction, and is required for activation of the helicase activity of RuvB. DNase I footprinting of the RuvAB branch migration motor assembled on a synthetic Holliday junction shows that two of the four DNA arms are bound by a RuvB hexamer (4Hiom K West S.C Cell. 1995; 80: 787-793Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Assuming that RuvB was a typical translocating helicase, a model for branch migration was proposed in which the RuvAB complex would move along the two recombining duplexes (Figure 4B). However, when recombination intermediates bound by RuvAB were visualized by electron microscopy (Figure 4C), the RuvB rings were found to be diametrically opposed across the RuvA-bound junction (10Parsons C.A Stasiak A Bennett R.J West S.C Nature. 1995; 374: 375-378Crossref PubMed Scopus (148) Google Scholar). How then do these two helicases promote branch migration? Two key observations suggested an answer: first, it was shown that binding of the Holliday junction by RuvA caused the DNA to unfold into an open square configuration; second, preliminary image analysis indicated that the two hexamers lie in opposite orientations across the junction (Figure 4D). The helicases therefore will exert equal and opposite forces to the DNA. Since the RuvB rings are tethered to the Holliday junction by RuvA and their opposing activities effectively neutralize any possibility of translocation along DNA anyway, the most plausible explanation is that the DNA is pulled into and through the protein complex. Thus, two helicases have been coupled to act as a “molecular pump” that drives strand exchange (Figure 4D). The difference between a “translocating motor” and a molecular pump might be more one of semantics than mechanism, since both promote the passage of DNA through the central cavity of a ring structure. In the case of RuvB, it is known that both DNA strands pass through the ring. Given the similarity in structure of RuvB to other hexameric helicases, it is possible that other ring proteins act by a similar helicase mechanism (although Rho may be an exception). If this is true, how are the two strands unwound, and how do the hexamer rings promote passage of DNA through the protein? Studies with DnaB and T7 gp4 indicate that a single strand of DNA is bound by one of the six subunits, and it is possible that this strand is passed sequentially from one subunit to the next by a reaction that involves cycles of ATP binding and hydrolysis. However, not all subunits within the hexamer are equivalent with respect to ATP hydrolysis, so the cycle may not involve every subunit. Indeed, each pair of subunits might function as a dimer within the ring hexamer. A mechanism that involves only one of the two DNA strands would provide the reaction with a defined polarity, but the position of the complementary DNA strand within the hexamer is unclear. Indeed, in the case of T7 gp4 and DnaB, it is possible that the second strand lies outside of the ring structure. Although the molecular pump action of RuvB is unusual, it is unlikely that the use of “two opposing helicases” is unique to recombination in E. coli. In fact, a similar reaction may occur at the initiation of replication of SV40 DNA by the viral-encoded large T antigen, another hexameric ring helicase (10Parsons C.A Stasiak A Bennett R.J West S.C Nature. 1995; 374: 375-378Crossref PubMed Scopus (148) Google Scholar). Because the origin is palindromic (i.e., two identical DNA sequences lie in opposite orientations), the two rings are likely to lie in opposite orientations. Here, dual helicase action (3′–5′) will lead to the unwinding of origin DNA to release template single strands at the double hexamer, as observed by electron microscopy (12Wessel R Schwizer J Stahl H J. Virol. 1992; 66: 804-815Crossref PubMed Google Scholar). Undoubtedly, we have much to learn about the way in which hexameric ring helicases translocate along DNA or promote the passage of DNA through a tethered protein machine. In future studies, it will be important to understand the detailed energetics of the translocation process, and toward this goal, we should be able to benefit from recent work with classical motor proteins such as myosin and kinesin (7Kull F.J Sablin E.P Lau R Fletterick R.J Vale R.D Nature. 1996; 380: 550-555Crossref PubMed Scopus (570) Google Scholar). Low-resolution structural analyses, such as those provided by electron microscopy, are beginning to provide clues to the function and mechanism of action of these remarkable proteins, but the field awaits the necessary breakthrough that can only be provided when the structure of a DNA helicase is solved by X-ray crystallography." @default.
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• W2000270965 title "DNA Helicases: New Breeds of Translocating Motors and Molecular Pumps" @default.
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