Polymerases and the Replisome: Machines within Machines
1998; Cell Press; Volume: 92; Issue: 3 Linguagem: Inglês
10.1016/s0092-8674(00)80923-x
ISSN1097-4172
AutoresTania A. Baker, Stephen P. Bell,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoSynthesis of all genomic DNA involves the highly coordinated action of multiple polypeptides. These proteins assemble two new DNA chains at a remarkable pace, approaching 1000 nucleotides (nt) per second in E. coli. If the DNA duplex were 1 m in diameter, then the following statements would roughly describe E. coli replication. The fork would move at approximately 600 km/hr (375 mph), and the replication machinery would be about the size of a FedEx delivery truck. Replicating the E. coli genome would be a 40 min, 400 km (250 mile) trip for two such machines, which would, on average make an error only once every 170 km (106 miles). The mechanical prowess of this complex is even more impressive given that it synthesizes two chains simultaneously as it moves. Although one strand is synthesized in the same direction as the fork is moving, the other chain (the lagging strand) is synthesized in a piecemeal fashion (as Okazaki fragments) and in the opposite direction of overall fork movement. As a result, about once a second one delivery person (i.e., polymerase active site) associated with the truck must take a detour, coming off and then rejoining its template DNA strand, to synthesize the 0.2 km (0.13 mile) fragments. In this review we describe our current understanding of the organization and function of the proteins of the replication fork and how these complexes are assembled at origins of replication. Understanding the architecture of DNA polymerases is relevant to RNA polymerases as well, as the core of the polynucleotide polymerization machine appears to be similar for all such enzymes. In the discussion of the replisome, we particularly focus on features shared by the machinery from different organisms. The replication fork contains several key activities that can be considered as machines on their own: (1) the specialized polymerases that synthesize new strands; (2) the editing exonuclease associated with the replicative polymerase; (3) the accessory proteins that control interaction of the polymerases with the DNA, and (4) the helicase that melts the DNA double helix to generate the replication fork. These components are functionally conserved in diverse organisms. Table 1 lists the replication proteins that serve similar functions from phage T4, E. coli, yeast and human cells (based on the requirements to replicate the SV40 virus). Below, we first outline the recent progress in understanding the activities, architecture, and mechanism of these component machines followed by a discussion of how they communicate with one another within the replisome.Table 1Proteins that Perform Analogous Functions at Replication ForksFunctionE. coli/Phage λPhage T4SV40/HumanYeastHelicaseDnaBgp41T antigen (SV40 specific)MCM proteins?PrimaseDnaG primasegp61Primase subunit of pol α–primasePrimase subunit of pol α–primasePolymeraseα subunit of DNA pol III H.E.gp43pol δpol δ and pol ε both involvedProofreading exonucleaseε subunit of DNA pol III H.E.Part of gp43 polymerase subunitPart of polymerase subunit of pol δPart of polymerase subunit of both pol ε and pol δSliding clampβ subunitgp45PCNAPCNAClamp loaderγ complexgp44/62RF-CRF-CSingle-strand DNA-binding proteinSSBgp32RP-ARP-AH.E., holoenzyme. Open table in a new tab H.E., holoenzyme. Synthesis of the new DNA strands occurs as a result of a collaboration between the synthetic capacities of multiple polymerases. Two types of polymerases are required: primases, which start chains, and replicative polymerases, which synthesize the majority of the DNA (37Kornberg A Baker T.A DNA Replication, Second Ed. W.H. Freeman and Company, New York1992Google Scholar). The replication fork, however, contains at least three distinct polymerase activities: a primase and a replicative polymerase for each of the two template strands. In E. coli, primase is a single polypeptide, and the replicative polymerase is a dimer of DNA polymerase (pol) III core and several accessory proteins that together form the pol III holoenzyme (reviewed in49Marians K.J Prokaryotic DNA replication.Annu. Rev. Biochem. 1992; 61: 673-719Crossref PubMed Google Scholar, 32Kelman Z O'Donnell M DNA polymerase III holoenzyme structure and function of a chromosomal replicating machine.Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (353) Google Scholar). Similarly, phage T4 has one primase and one replicative polymerase that appears to function as a dimer (1Alberts B.M Prokaryotic DNA replication mechanisms.Philos. Trans. R. Soc. Lond. B. 1987; 317: 395-420Crossref PubMed Scopus (66) Google Scholar, 52Munn M.M Alberts B.M The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction.J. Biol. Chem. 1991; 266: 20024-20033Abstract Full Text PDF PubMed Google Scholar). The situation in eukaryotic cells is slightly different (68Stillman B Smart machines at the DNA replication fork.Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (219) Google Scholar). The primase is in a tight complex with a DNA polymerase (pol α) and eukaryotic cells have two distinct replicative polymerases: polymerase δ (pol δ) and polymerase ε (pol ε). All the replicative polymerases have one large subunit that contains the polymerase active site and, with the exception of pol α–primase, the same subunit or an associated polypeptide carries a proofreading 3′→5′ exonuclease. The polymerase subunits also interact with proteins that dramatically influence their association with DNA. In E. coli, the replicative polymerase is found in a complex with proteins that control polymerase processivity; this holoenzyme, consists of 10 distinct polypeptides (32Kelman Z O'Donnell M DNA polymerase III holoenzyme structure and function of a chromosomal replicating machine.Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (353) Google Scholar). In contrast, neither the T4 nor the eukaryotic polymerases copurify in a complex with the processivity factors (1Alberts B.M Prokaryotic DNA replication mechanisms.Philos. Trans. R. Soc. Lond. B. 1987; 317: 395-420Crossref PubMed Scopus (66) Google Scholar, 68Stillman B Smart machines at the DNA replication fork.Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (219) Google Scholar). Therefore, these proteins are called accessory proteins rather than subunits (see Table 1). Polymerase Architecture. The central feature of all the known polymerase structures is the existence of a large cleft comprised of three subdomains referred to as the fingers, palm, and thumb by virtue of the similarity of the structures to a half-opened right hand (Figure 1; polymerase structures are reviewed in 29Joyce C.M Steitz T.A Function and structure relationships in DNA polymerases.Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (553) Google Scholar, 30Joyce C.M Steitz T.A Polymerase structures and function variations on a theme?.J. Bacteriol. 1995; 177: 6321-6329Crossref PubMed Google Scholar, 65Sousa R Structural and mechanistic relationships between nucleic acid polymerases.Trends Biochem. Sci. 1996; 21: 186-190Abstract Full Text PDF PubMed Scopus (139) Google Scholar). A diverse set of polymerases—including several replicative and repair DNA polymerases from viral, prokaryotic, and eukaryotic sources, reverse transcriptase, and even an RNA polymerase—share this general structure. The palm subdomain, at the bottom of the cleft, contains the active site, including the essential acidic amino acids that bind metal ions involved in catalysis, residues that interact with the primer terminus, and the α-phosphate of the incoming dNTP. The conserved amino acid sequence motifs A and C, found in all nucleic acid polymerases, and motif B, found in the DNA-dependent enzymes, are present within this palm domain where they contribute to the active site (Figure 1A). The walls of the polymerase cleft are made up of the finger and thumb subdomains. Although less well conserved than the catalytic palm (in some polymerases, these domains are unrelated), these subdomains contribute analogous functions in many polymerases (80Wang J Sattar A.K Wang C.C Karam J.D Konigsberg W.H Steitz T.A Crystal structure of a pol α family replication DNA polymerase from bacteriophage RB69.Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 33Kiefer J.R Mao C Braman J.C Beese L.S Visualizing DNA replication in a catalytically active polymerase crystal at 1.8 angstrom resolution.Nature. 1998; in pressGoogle Scholar; and reviewed in 30Joyce C.M Steitz T.A Polymerase structures and function variations on a theme?.J. Bacteriol. 1995; 177: 6321-6329Crossref PubMed Google Scholar, 65Sousa R Structural and mechanistic relationships between nucleic acid polymerases.Trends Biochem. Sci. 1996; 21: 186-190Abstract Full Text PDF PubMed Scopus (139) Google Scholar). Based on DNA cocrystal structures and modeling studies, the fingers subdomain makes contact with the single-stranded template strand, that has yet to be copied. Part of the fingers domain, along with the palm domain, is involved in binding the incoming substrate dNTP. The thumb subdomain interacts with the template-primer DNA helix (Figure 1B). The recently solved structure of the B. stearothermophilus DNA pol I (49% identical in sequence to the E. coli enzyme) with a primer-template in the polymerase active site, provides insight into the mechanism by which polymerases interact with DNA in a sequence-independent manner (33Kiefer J.R Mao C Braman J.C Beese L.S Visualizing DNA replication in a catalytically active polymerase crystal at 1.8 angstrom resolution.Nature. 1998; in pressGoogle Scholar). The polymerase makes extensive interactions with the DNA minor groove of the first four base pairs (with respect to the 3′ primer terminus) of the primer-template helix. Minor groove contacts allow binding to any sequence because the minor groove, in contrast to the major groove, contains a pattern of hydrogen bond donors and acceptors that are independent of the nucleotide sequence, as long as the bases are in proper Watson-Crick pairs. A central feature of polymerase active sites is the cluster of conserved carboxylates and other polar residues at the base of the cleft in the palm domain (67Steitz T.A Smerdon S.J Jager J Joyce C.M A unified polymerase mechanism for nonhomologous DNA and RNA polymerases.Science. 1994; 266: 2022-2025Crossref PubMed Scopus (266) Google Scholar). These carboxylates anchor two divalent metal ions involved in catalysis. The polymerization reaction proceeds by nucleophilic attack by the 3′ hydroxyl of the primer terminus on the dNTP α-phosphate with release of PPi. One divalent metal ion is thought to promote the deprotonation of the 3′ hydroxyl of the primer strand whereas the other facilitates formation of the pentacovalent transition state at the α-phosphate of the dNTP and the departure of the PPi leaving group. Similar two-metal mechanisms have been proposed to catalyze phosphoryl transfer reactions in numerous other systems including the proofreading exonuclease associated with polymerases and RNaseH domains associated with reverse transcriptases (29Joyce C.M Steitz T.A Function and structure relationships in DNA polymerases.Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (553) Google Scholar). In addition to the acidic amino acids from the palm subdomain, several residues from the fingers subdomain participate directly in catalysis in the pol I family of polymerases. For example, a tyrosine residue in the B. stearothermophilus DNA pol I plays a critical function in establishing the geometry of the active site, thereby enforcing the requirement for proper base pairing prior to catalysis (33Kiefer J.R Mao C Braman J.C Beese L.S Visualizing DNA replication in a catalytically active polymerase crystal at 1.8 angstrom resolution.Nature. 1998; in pressGoogle Scholar). The close similarity between the structures of DNA pol I and the T7 RNA polymerase clearly indicate that these two proteins arose from a common ancestor (reviewed in30Joyce C.M Steitz T.A Polymerase structures and function variations on a theme?.J. Bacteriol. 1995; 177: 6321-6329Crossref PubMed Google Scholar). Furthermore, the palm subdomains of the pol α family polymerase (RB69 gp43), E. coli pol I and HIV reverse transcriptase can all be superimposed (80Wang J Sattar A.K Wang C.C Karam J.D Konigsberg W.H Steitz T.A Crystal structure of a pol α family replication DNA polymerase from bacteriophage RB69.Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). In contrast, the mammalian DNA pol β is distinct, being more similar to the nucleotidyl transferase enzyme family, and it has been argued that the similarities between this protein and the other polymerases are an example of convergent evolution. Comparisons of the structures and sequence motifs present in different polymerases also provide clues to the molecular mechanisms determining the specificity of different family members. For example, specific motifs found in DNA polymerases but not in RNA polymerases correlate with the specificity for dNTPs versus rNTPs and the requirement for a primer (65Sousa R Structural and mechanistic relationships between nucleic acid polymerases.Trends Biochem. Sci. 1996; 21: 186-190Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 28Joyce C.M Choosing the right sugar how polymerases select a nucleotide substrate.Proc. Natl. Acad. Sci. USA. 1997; 94: 1619-1622Crossref PubMed Scopus (181) Google Scholar). Editing. The polymerases responsible for the majority of DNA synthesis in phage, prokaryotes, and eukaryotes (phage T4 gp43, DNA pol III holoenzyme, pol δ and pol ε) all have an associated proofreading exonuclease. These activities, which preferentially excise a mismatched nucleotide from the primer terminus, contribute about three orders of magnitude to the fidelity of DNA replication (37Kornberg A Baker T.A DNA Replication, Second Ed. W.H. Freeman and Company, New York1992Google Scholar). The central features of this editing mechanism are likely to be general as many polymerases carry exonuclease domains that are similar in amino acid sequence. Sequence alignments, structural studies, and site-directed mutagenesis indicate that the exonuclease active site and the polymerase active site of these enzymes can be considered largely independent catalytic modules (29Joyce C.M Steitz T.A Function and structure relationships in DNA polymerases.Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (553) Google Scholar). The mechanism of editing is most thoroughly understood for E. coli DNA pol I (24Freemont P.S Friedman J.M Beese L.S Sanderson M.R Steitz T.A Cocrystal structure of an editing complex of Klenow fragment with DNA.Proc. Natl. Acad. Sci. USA. 1988; 85: 8924-8928Crossref PubMed Scopus (317) Google Scholar, 29Joyce C.M Steitz T.A Function and structure relationships in DNA polymerases.Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (553) Google Scholar). Its polymerase and exonuclease active centers are located 30 Å apart but are linked by a shared DNA binding cleft. This arrangement dictates that the 3′ end of the growing chain must switch from the polymerase active site to the editing active site for a mistake to be excised (Figure 1B). This switch reflects a preference of the polymerase active site for a properly base-paired primer terminus; a misincorpation results in a 3′ terminus that is not base paired and therefore slows the forward rate of polymerization. This misincorporation also promotes melting of the primer-template duplex to generate the preferred substrate for the exonuclease, a DNA molecule with the last 4–5 nt at the 3′ end single stranded. Thus, incorporation of a mismatched base simultaneously encourages melting of the duplex to generate the substrate for the exonuclease while inhibiting the polymerase. Processivity Factors: Sliding Clamps and Clamp Loaders. The exceptional processivity of replicative polymerases is controlled by protein subunits specialized for this function. The replicative polymerases of phage T4, E. coli, and eukaryotic cells each have two key processivity factors: (1) the sliding clamp and (2) the clamp loader (83Yao N Turner J Kelman Z Stukenberg P.T Dean F Shechter D Pan Z.Q Hurwitz J O'Donnell M Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.Genes Cells. 1996; 1: 101-113Crossref PubMed Scopus (177) Google Scholar). Sliding clamps are protein rings that encircle the DNA (36Kong X.P Onrust R O'Donnell M Kuriyan J Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme a sliding DNA clamp.Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 40Krishna T.S Kong X.P Gary S Burgers P.M Kuriyan J Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (725) Google Scholar). Examples of sliding clamps include phage T4 gp45, the E. coli β subunit of DNA pol III holoenzyme, and the eukaryotic PCNA. The different clamp proteins, although distinct in sequence and multimeric state (a "ring" of β is a dimer, whereas PCNA is a trimer) have very similar folds (36Kong X.P Onrust R O'Donnell M Kuriyan J Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme a sliding DNA clamp.Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 40Krishna T.S Kong X.P Gary S Burgers P.M Kuriyan J Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (725) Google Scholar). The structures of both the β subunit and PCNA reveal that each protein is a doughnut-shaped multimer, with a 35 Å hole, big enough for a duplex DNA to slide through the middle without physically contacting the protein; indeed there is room for one to two layers of water molecules between the inner protein surface and the DNA, which may facilitate sliding (36Kong X.P Onrust R O'Donnell M Kuriyan J Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme a sliding DNA clamp.Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 40Krishna T.S Kong X.P Gary S Burgers P.M Kuriyan J Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (725) Google Scholar). These clamp proteins are topologically linked to, rather than in physical contact with, the DNA (41Kuriyan J O'Donnell M Sliding clamps of DNA polymerases.J. Mol. Biol. 1993; 234: 915-925Crossref PubMed Scopus (198) Google Scholar). As a result of this mode of DNA interaction, clamp proteins remain stably bound to a circular DNA molecule but rapidly dissociate from the same DNA once it is linearized; dissociation occurs upon DNA cleavage because the protein simply slides off the end of the DNA. By interacting with the polymerase while remaining linked around the DNA, these proteins clamp the enzymatic subunits to the template (70Stukenberg P.T Turner J O'Donnell M An explanation for lagging strand replication polymerase hopping among DNA sliding clamps.Cell. 1994; 78: 877-887Abstract Full Text PDF PubMed Scopus (144) Google Scholar). Because the sliding clamps are closed circles of protein, energy-dependent clamp-loader machines are needed to assemble them onto DNA (Figure 2). The clamp loaders of phage T4 (gp44/62), E. coli (the γ complex), and eukaryotic cells (RF-C) each consist of multiple subunits, some of which are DNA-dependent ATPases (54O'Donnell M Onrust R Dean F.B Chen M Hurwitz J Homology in accessory proteins of replicative polymerases—E. coli to humans.Nucleic Acids Res. 1993; 21: 1-3Crossref PubMed Scopus (140) Google Scholar). The basic steps involved in loading include: recognition of the primer-template junction, binding the sliding clamp, disruption of the subunit interactions to open the ring, and placement of the ring around the DNA near a primer terminus. The following series of steps have been proposed for the mechanism of clamp loading by the E. coli γ complex (32Kelman Z O'Donnell M DNA polymerase III holoenzyme structure and function of a chromosomal replicating machine.Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (353) Google Scholar): (1) the γ complex binds ATP and undergoes a conformational change to expose the β-binding surface of the γ complex's δ subunit; (2) the δ subunit binds β and opens the β ring; (3) the γ complex then recognizes the primer-template DNA and brings β to the DNA; (4) ATP-hydrolysis or ADP release then reburies the δ subunit in the complex, destablizing the δ–β interaction, thereby causing the β subunit to "snap" shut around the DNA. Interestingly, the γ complex can promote both the loading and unloading of β rings from the DNA. Whether interaction of γ complex with β results in loading or unloading is modulated by the interaction between β and the α subunit of pol III holoenzyme (53Naktinis V Turner J O'Donnell M A molecular switch in a replication machine defined by an internal competition for protein rings.Cell. 1996; 84: 137-145Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Because polymerases and the clamp loader interact with the same face of the clamp, β subunits that are complexed with a polymerase are specifically protected from unloading, whereas those free from a polymerase may be unloaded and recycled. Evidence for similar loading schemes has emerged from studies of the T4 and eukaryotic clamp loaders (76Tsurimoto T Stillman B Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins.J. Biol. 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Sequence homology and structure-based alignments indicate that the clamp loader subunits are a family of related proteins that are likely to have similar folds (25Guenther B Onrust R Sali A O'Donnell M Kuriyan J Crystal structure of the δ′ subunit of the clamp loader complex of E. coli DNA polymerase III.Cell. 1997; 91: 335-346Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The crystal structure of one of the clamp loader subunits, the δ' protein of the γ complex, reveals that it is a "C"-shaped protein (25Guenther B Onrust R Sali A O'Donnell M Kuriyan J Crystal structure of the δ′ subunit of the clamp loader complex of E. coli DNA polymerase III.Cell. 1997; 91: 335-346Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). The location of the ATP-binding site in these proteins is positioned such that ATP binding or hydrolysis could cause a conformational change; that this change results in the mouth of the "C" cycling between open and closed states is an attractive model for a protein that must open protein clamps using the energy of ATP hydrolysis. Primases. Several features distinguish primases from replicative polymerases. Primases are unique among the polymerases involved in DNA replication in their ability to start the synthesis of new polynucleotide chains (37Kornberg A Baker T.A DNA Replication, Second Ed. W.H. Freeman and Company, New York1992Google Scholar). Primases initiate chain synthesis at preferred sites on the template DNA; these "start sites" correspond to degenerate trinucleotide sequences (37Kornberg A Baker T.A DNA Replication, Second Ed. W.H. Freeman and Company, New York1992Google Scholar). Thus, there are many places where primers can be initiated. Nonetheless, this sequence preference clearly distinguishes primases from replicative DNA polymerases. Different primases recognize different sequences. In some primases a zinc finger–like DNA-binding domain is involved in DNA sequence selection (42Kusakabe T Richardson C.C The role of the zinc motif in sequence recognition by DNA primases.J. Biol. Chem. 1996; 271: 19563-19570Crossref PubMed Scopus (57) Google Scholar). Most primases can use either deoxy- or ribonucleotides; however, primers are usually RNA because of the larger cellular pools of ribonucleotides. Primases have very limited processivity and usually synthesize chains shorter than 12 nt. In eukaryotic cells, RNA primers are synthesized by the bifunctional pol α–primase and the short RNA primers synthesized by the primase active site are rapidly elongated by the associated DNA polymerase (see below). The familiar structure of a replication fork as a site where the two strands of a duplex DNA are separated to reveal the single strands of opposite polarity, is generated through the action of a replicative DNA helicase. Although RNA polymerases can melt a DNA duplex, replicative DNA polymerases depend on a separate helicase. Helicases, or proteins with sequence homology to helicases, have been discovered with functions in DNA repair, genetic recombination, or transcription, however, only a subset of helicases are specialized to create replication forks. Recent work on the E. coli replicative helicase DnaB, the SV40 T antigen, and the phage helicases from T7 and T4, reveal that these four proteins have a common hexameric architecture and similar biochemical properties including high processivity and synergistic interactions with their cognate replicative DNA polymerases (23Egelman E.H Homomorphous hexameric helicases tales from the ring cycle.Structure. 1996; 4: 759-762Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, see below). Even in the absence of a clear sequence relationship, it is likely that these properties will be widespread among helicases that generate replication forks. Electron microscopy image reconstruction techniques reveal that the hexameric replicative helicases form protein rings that can encircle DNA. The phage T7 helicase, for example, is a hexameric ring with two distinct faces (C6 symmetry, 85Yu X Hingorani M.M Patel S.S Egelman E.H DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase.Nat. Struct. Biol. 1996; 3 (a): 740-743Crossref PubMed Scopus (113) Google Scholar). Single-stranded DNA passes through the center of this protein ring; a similar arrangement is thought to exist for the DnaB, the T4 helicase and the SV40 T antigen–DNA complexes (23Egelman E.H Homomorphous hexameric helicases tales from the ring cycle.Structure. 1996; 4: 759-762Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Whether one or both strands of DNA enter the ring in each case is not yet clear and may differ among the different helicases. The T7 helicase ring is 130 Å in diameter with a 25 to 30 Å hole and covers about 30 nt of DNA (85Yu X Hingorani M.M Patel S.S Egelman E.H DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase.Nat. Struct. Biol. 1996; 3 (a): 740-743Crossref PubMed Scopus (113) Google Scholar). The fact that these helicases can encircle DNA provides a structural explanation for their nearly unlimited processivity in the context of a replication fork. Thus, both the sliding clamps and the hexameric DNA helicases appear to have met the requirement for high processivity by becoming topologically linked to the DNA. Once these helicases associate productively with DNA, helix melting continues until some active process terminates helicase activity. Sequence-specific termination proteins provide such helicase road blocks in bacteria (reviewed in4Baker T.A Replication arrest.Cell. 1995; 80: 521-524Abstract Full Text PDF PubMed Scopus (27) Google Scholar). How ATP (NTP) fuels unwinding by the hexameric DNA helicases is not yet clear; however, some basic features of the cycle are emerging (see48Lohman T.M Bjornson K.P Mechanisms of helicase-catalyzed DNA unwinding.Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (650) Google Scholar, for a recent review of helicase mechanism). The helicase cycle must involve an ordered series of conformational changes, modulated by ATP (NTP) binding, hydrolysis and release, allowing it to move along the DNA (50Marians K.J Helicase structures a new twist on DNA unwinding.Structure. 1997; 5: 1129-1134Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). For example, one conformation may interact with the duplex DNA, whereas a second conformation binds the melted single strands; ATP-dependent switching between these conformations may therefore represent the "power stroke" that melts the duplex and propels the helicase forward on the DNA strand. The existence of distinct conformational states of the hexameric helicases is supported by structural and kinetic studies (9Bujalowski W Klonowska M.M Jezewska M.J Oligomeric structure of Escherichia coli primary replicative helicase DnaB protein.J. Biol. Chem. 1994; 269: 31350-31358Abstract Full Text PDF PubMed Google Scholar, 60San Martin M Stamford N.P Dammerova N Dixon N.E Carazo J.M A structural model for the Escherichia coli DnaB helicase based on electron microscopy data.J. Struct. Biol. 1995; 114: 167-176Crossref PubMed Scopus (130) Goo
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