Coordination between the Polymerase and 5′-Nuclease Components of DNA Polymerase I of Escherichia coli
2000; Elsevier BV; Volume: 275; Issue: 27 Linguagem: Inglês
10.1074/jbc.m909135199
ISSN1083-351X
AutoresYang Xu, Nigel D. F. Grindley, Catherine M. Joyce,
Tópico(s)DNA Repair Mechanisms
ResumoThe polymerase and 5′-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5′-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5′-nuclease. The preferred substrate of the 5′-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5′-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5′-nuclease activities are coupled such that the majority of molecules cleaved by the 5′-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5′-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution. The polymerase and 5′-nuclease components of DNA polymerase I must collaborate in vivo so as to generate ligatable structures. Footprinting shows that the polymerase and 5′-nuclease cannot bind simultaneously to a DNA substrate and appear to compete with one another, suggesting that the two active sites are physically separate and operate independently. The desired biological end point, a ligatable nick, results from the substrate specificities of the polymerase and 5′-nuclease. The preferred substrate of the 5′-nuclease is a "double-flap" structure having a frayed base at the primer terminus overlapping the displaced strand that is to be cleaved by the 5′-nuclease. Cleavage of this structure occurs almost exclusively between the first two paired bases of the downstream strand, yielding a ligatable nick. In whole DNA polymerase I, the polymerase and 5′-nuclease activities are coupled such that the majority of molecules cleaved by the 5′-nuclease have also undergone polymerase-catalyzed addition to the primer terminus. This implies that the 5′-nuclease can capture a DNA molecule from the polymerase site more efficiently than from the bulk solution. DNA polymerase I The DNA polymerase I (Pol I)1 enzymes of eubacteria function in DNA repair and in the removal of RNA primers from Okazaki fragments during lagging strand replication (1.Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Co., San Francisco1992Google Scholar). To facilitate the formation of ligatable structures, the bacterial Pol I enzymes usually have nuclease activity, which degrades the downstream DNA or RNA strand. Originally described as a 5′-3′ exonuclease, this activity is now recognized to be a structure-specific nuclease with specificity for the junction between a DNA duplex and a 5′-single-stranded tail (or flap) and is therefore better described as a 5′-nuclease (2.Lyamichev V. Brow M.A.D. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (302) Google Scholar). On a nicked DNA duplex, polymerase-catalyzed primer extension continuously regenerates the substrate for the 5′-nuclease, so that the polymerase effectively drives nick translation (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar). The 5′-nuclease activity of Pol I is present on an independent structural domain encoded by the first one-third of the structural gene, which can be separated from the polymerase by proteolysis or by recombinant DNA manipulations (4.Klenow H. Overgaard-Hansen K. FEBS Lett. 1970; 6: 25-27Crossref PubMed Scopus (54) Google Scholar, 5.Setlow P. Kornberg A. J. Biol. Chem. 1972; 247: 232-240Abstract Full Text PDF PubMed Google Scholar, 6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar). The structure specificity is inherent to the 5′-nuclease domain itself and does not rely on the presence of the polymerase domain (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar, 7.Lyamichev V. Brow M.A.D. Varvel V.E. Dahlberg J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6143-6148Crossref PubMed Scopus (63) Google Scholar). Indeed, some bacteriophages encode separate nucleases, having a high degree of homology to the N-terminal region of the bacterial Pol I enzymes, which act in concert with the relevant bacteriophage polymerase (8.Sayers J.R. Eckstein F. J. Biol. Chem. 1990; 265: 18311-18317Abstract Full Text PDF PubMed Google Scholar, 9.Hollingsworth H.C. Nossal N.G. J. Biol. Chem. 1991; 266: 1888-1897Abstract Full Text PDF PubMed Google Scholar, 10.Gutman P.D. Minton K.W. Nucleic Acids Res. 1993; 21: 4406-4407Crossref PubMed Scopus (54) Google Scholar). In eukaryotes and archaebacteria, the structure-specific cleavages required in replication and repair are carried out by "flap endonucleases" (11.Lieber M.R. Bioessays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar, 12.Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 13.Shen B. Qiu J. Hosfield D. Tainer J.A. Trends. Biochem. Sci. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), which exist as independent protein subunits and are therefore able to collaborate with a variety of polymerases. The flap endonuclease family of proteins shows only a modest level of sequence similarity to the bacterial and bacteriophage 5′-nucleases (14.Mueser T.C. Nossal N.G. Hyde C.C. Cell. 1996; 85: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), but the similarity in three-dimensional structures (14.Mueser T.C. Nossal N.G. Hyde C.C. Cell. 1996; 85: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15.Kim Y. Eom S.H. Wang J. Lee D.-S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (328) Google Scholar, 16.Ceska T.A. Sayers J.R. Stier G. Sück D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (164) Google Scholar, 17.Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (147) Google Scholar, 18.Hosfield D.J. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) and in the reactions carried out by these two families of 5′-nucleases leaves little doubt that they are functionally analogous to one another.In vivo, the polymerase and 5′-nuclease of bacterial Pol I must collaborate so as to leave a nick that can be sealed by DNA ligase. Two extreme scenarios can be envisaged. The Pol I molecule might adopt a structure that brings the two active sites into close proximity so they can bind simultaneously to the DNA substrate. Alternatively, the polymerase and 5′-nuclease activities might operate essentially independently of one another, perhaps even with the DNA traveling from one active site to the other via dissociation into free solution. This latter scenario fits well with the existence of a separate 5′-nuclease in some systems and is analogous to the relationship between the polymerase and 3′-5′ exonuclease (editing) functions of Klenow fragment, where the two active sites are separated by about 30 Å, with a substantial amount of the transfer between them occurring via dissociation (19.Joyce C.M. J. Biol. Chem. 1989; 264: 10858-10866Abstract Full Text PDF PubMed Google Scholar). Close juxtaposition of polymerase and 5′-nuclease active sites might be difficult to achieve; the polymerase active site is located at the base of a cleft (20.Brautigam C.A. Steitz T.A. Curr. Opin. Struct. Biol. 1998; 8: 54-63Crossref PubMed Scopus (334) Google Scholar), and the 5′-nuclease domain is structurally complex, possibly requiring threading of the unpaired 5′ end through an arch or loop of the protein (16.Ceska T.A. Sayers J.R. Stier G. Sück D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (164) Google Scholar, 17.Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (147) Google Scholar, 18.Hosfield D.J. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). However, recent structures of polymerases with DNA bound at the polymerase site suggest a dislocation of the downstream DNA template beyond the site of synthesis (21.Doublié S. Tabor S. Long A. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 22.Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar), and such a dislocation might provide a way to accommodate both polymerase and 5′-nuclease active sites in reasonably close proximity. Only two polymerase crystal structures (both of Taq DNA polymerase) include a 5′-nuclease domain. The first structure showed the polymerase and 5′-nuclease sites separated by about 70 Å at opposite ends of a rather extended molecule, arguing in favor of separate and independent active sites; however, x-ray scattering measurements suggested that the polymerase may fold more compactly in solution (15.Kim Y. Eom S.H. Wang J. Lee D.-S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (328) Google Scholar). A more recent crystal structure of Taq DNA polymerase complexed with an Fab showed the nuclease domain (which was remote from the Fab location) close to the fingers subdomain of the polymerase (23.Murali R. Sharkey D.J. Daiss J.L. Murthy H.M.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12562-12567Crossref PubMed Scopus (29) Google Scholar). Although the active sites were still separated by almost 40 Å, this location brings the 5′-nuclease much closer to the downstream portion of a DNA molecule bound at the polymerase active site. At the very least, the structural data suggest a flexible linkage between the 5′-nuclease and the rest of the polymerase molecule, and this could allow the two domains to be closely associated in an active complex.In this work, we have investigated two aspects of the collaboration between the polymerase and 5′-nuclease components of Escherichia coli Pol I: the way in which the substrate preferences of both activities are biased so as to produce a ligatable nick and the extent to which polymerase and 5′-nuclease are coupled so that both reactions take place within the same DNA binding event.DISCUSSIONTo perform its functions in vivo, in excision repair and in lagging strand replication, Pol I must leave a ligatable nick. Achieving this end point requires a delicate balance between polymerase and 5′-nuclease activities; an imbalance will give either a gapped or a 5′-tailed duplex, neither of which is a substrate for DNA ligase. Our results imply that the DNA substrate cannot contact both polymerase and 5′-nuclease active sites simultaneously but, rather, must be passed back and forth between two autonomous and non-overlapping active sites. The correct end point, a ligatable nick, results from the substrate preferences of the two domains and the cleavage specificity of the 5′-nuclease.Polymerase and 5′-Nuclease Sites Are Separate and Operate IndependentlyOur footprinting data show that the polymerase and 5′-nuclease domains, when present on separate molecules, cannot bind simultaneously to a DNA substrate. The slight overlap between the footprints of the separate domains accounts for the inhibition by Klenow fragment of both the binding and nuclease activity of the 5′-nuclease domain. The substrate specificities described below imply that the polymerase senses what is beyond the primer terminus and the 5′-nuclease senses the location of the upstream primer strand, and this fits with the observed footprints. When the polymerase and 5′-nuclease are covalently joined in whole Pol I, the polymerase mode of binding dominates, consistent with its greater DNA binding affinity (6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar, 27.Polesky A.H. Steitz T.A. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1990; 265: 14579-14591Abstract Full Text PDF PubMed Google Scholar,29.Astatke M. Grindley N.D.F. Joyce C.M. J. Biol. Chem. 1995; 270: 1945-1954Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Failure to observe a Pol I footprint that is the sum of the footprints of the two separate domains suggests that no benefit results from the high local concentration of the 5′-nuclease domain when the polymerase is bound to DNA and argues strongly that the two domains do not cooperate so as to bind simultaneously to the DNA substrate. The apparent antagonism between the polymerase and nuclease binding modes probably accounts for the lower activity of the 5′-nuclease when present in whole Pol I (6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar).Formation of a Ligatable NickBoth polymerase and 5′-nuclease discriminate between related substrate structures in such a way as to increase the probability of generating a ligatable nick. The preference of the polymerase for gapped structures helps to ensure that gaps are filled, whereas rapid dissociation from a nick allows other enzymes to act. The preferred substrate of the 5′-nuclease is a double-flap molecule with an unpaired base at the primer terminus. Cleavage of the double-flap DNA is focused almost exclusively to a single position between the first two paired bases of the strand with the 5′ overhang, generating a ligatable nick. The efficient processing of this substrate to yield the required product suggests that the double-flap structure may be the natural substrate for the 5′-nuclease. A preference for double-flap substrates has also been reported for the 5′-nucleases ofTaq and Tth DNA polymerases and for eukaryotic and archaebacterial FEN-1 enzymes (7.Lyamichev V. Brow M.A.D. Varvel V.E. Dahlberg J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6143-6148Crossref PubMed Scopus (63) Google Scholar, 30.Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 31.Kaiser M.W. Lyamicheva N. Ma W. Miller C. Neri B. Fors L. Lyamichev V. J. Biol. Chem. 1999; 274: 21387-21394Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).In contrast to 5′-nuclease cleavage of the double-flap structure, the reaction with nicked and gapped substrates seems much less efficient and precise. The predominant cleavage site on a nicked or gapped substrate is between the first two paired bases of the downstream strand (Fig. 7) (2.Lyamichev V. Brow M.A.D. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (302) Google Scholar), which means that cleavage must be followed by at least one more round of polymerase addition before ligation can take place. Cleavage of nicked and gapped substrates often occurs at more than one position, and this could reflect formation (via branch migration) of several interconvertible structures. The extent to which a particular cleavage site is represented in the reaction products would be determined by the abundance of the relevant structure and the efficiency with which it is cleaved by the 5′-nuclease. Specifically, we suggest that the cleavages that apparently map within the single-stranded 5′ tail actually involve rearrangement of the DNA to give double-flap structures, usually having imperfect base pairing around the cleavage site. These double-flap and related structures need not be abundant in the substrate pool if they are strongly preferred as nuclease substrates. Similar reasoning has been invoked to explain the formation of a variety of cleavage products by the Taq5′-nuclease (7.Lyamichev V. Brow M.A.D. Varvel V.E. Dahlberg J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6143-6148Crossref PubMed Scopus (63) Google Scholar). Examination of the structural model proposed for T5 5′-nuclease bound to its DNA substrate (16.Ceska T.A. Sayers J.R. Stier G. Sück D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (164) Google Scholar) suggests that there could be room for an additional base at the primer terminus; contacts between the active site and the 3′-unpaired base would then account for the preference of the 5′-nuclease for the double-flap substrate.The potential for 5′-nuclease substrates (particularly those made by strand-displacement synthesis) to rearrange and form double-flap structures may account for some inconsistencies in the cleavage sites reported for these enzymes. For example, Lundquist and Olivera (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar) consistently observed cleavage by the 5′-nuclease of E. coliPol I apparently at the junction between the downstream duplex and the 5′ single strand, whereas other studies agree that the predominant cleavage position for the bacterial Pol I 5′-nucleases is one base 3′ to this position, i.e. between the first two paired downstream bases (2.Lyamichev V. Brow M.A.D. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (302) Google Scholar, 6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar, 32.Kelly R.B. Atkinson M.R. Huberman J.A. Kornberg A. Nature. 1969; 224: 495-501Crossref Scopus (183) Google Scholar). Significantly, Lundquist and Olivera (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar) made their DNA substrates by polymerase-catalyzed primer extension, whereas the other studies used synthetic oligonucleotides in which the possibilities for branch migration were more limited.Our data suggest the following scenario for the processing of a DNA substrate by Pol I (Fig. 9). The polymerase extends the upstream primer strand, in some cases proceeding beyond the junction with the downstream DNA. Branch migration can then generate a family of interconvertible structures. Double-flap structures with a single frayed base at the primer terminus will be the most readily cleaved by the 5′-nuclease, generating a nick that discourages binding of the polymerase, allowing access of DNA ligase. Other conformations will lack the full complement of contacts to the 5′-nuclease domain and will therefore be cleaved less rapidly, allowing time for further rearrangement to a more optimal substrate or generating a product that can undergo additional cycles of extension and cleavage.Coupling of Polymerase and 5′-NucleaseWhen both polymerase and 5′-nuclease are covalently linked in whole Pol I, the fraction of product molecules that have undergone both reactions is greater than when polymerase and nuclease are on separate molecules, implying that both enzymatic reactions can take place within the same protein-DNA binding event. Given the greater binding affinity and reaction rate at the polymerase active site, the most likely scenario is that the polymerase acts first, giving a DNA intermediate that is then cleaved by the 5′-nuclease. Covalent linkage of the two domains delivers a high effective concentration of the 5′-nuclease, compensating for the rather weak binding of DNA to this domain. Nevertheless, the majority of the DNA molecules extended by the polymerase dissociate rather than become substrates for the 5′-nuclease, and this probably argues against an active mode of channeling the DNA intermediate from one active site to the other. Instead, coupling of the two activities may involve partial dissociation of the DNA from one active site followed by capture by the other active site before it is lost into the bulk solution.Polymerase activity is also coupled to variable extents to some of the other auxiliary functions present in polymerases. Coupling between the polymerase and 3′-5′-editing exonuclease activity is rather limited in Klenow fragment (19.Joyce C.M. J. Biol. Chem. 1989; 264: 10858-10866Abstract Full Text PDF PubMed Google Scholar) but greater in T4 and T7 DNA polymerases, where the 3′-5′ exonuclease reaction is more rapid and therefore competes more effectively with dissociation (34.Capson T.L. Peliska J.A. Kaboord B.F. Frey M.W. Lively C. Dahlberg M. Benkovic S.J. Biochemistry. 1992; 31: 10984-10994Crossref PubMed Scopus (225) Google Scholar, 35.Donlin M.J. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 538-546Crossref PubMed Scopus (179) Google Scholar). By contrast, there appears to be tight coupling between polymerase and RNase H activity in human immunodeficiency virus-1 reverse transcriptase (36.Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar). The important difference may be that in reverse transcriptase the nucleic acid substrate is able to contact both polymerase and RNase H sites simultaneously (37.Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1354) Google Scholar), whereas a DNA substrate has to transfer between the polymerase and editing sites of DNA polymerases. The coupling we have observed between polymerase and 5′-nuclease is not necessarily restricted to those systems in which the two activities are covalently linked. The activity of the bacteriophage T4 5′-nuclease (T4 RNase H) on the lagging strand may be integrated with the rest of the T4 replication complex through an interaction with the gene 32 single-stranded-binding protein (38.Bhagwat M. Hobbs L.J. Nossal N.G. J. Biol. Chem. 1997; 272: 28523-28530Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and the eukaryotic FEN-1 enzymes may be localized at the replication fork by association with the processivity factor, proliferating cell nuclear antigen (PCNA) (39.Wu X. Li J. Li X. Hsieh C.-L. Burgers P.M.J. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar). The DNA polymerase I (Pol I)1 enzymes of eubacteria function in DNA repair and in the removal of RNA primers from Okazaki fragments during lagging strand replication (1.Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman and Co., San Francisco1992Google Scholar). To facilitate the formation of ligatable structures, the bacterial Pol I enzymes usually have nuclease activity, which degrades the downstream DNA or RNA strand. Originally described as a 5′-3′ exonuclease, this activity is now recognized to be a structure-specific nuclease with specificity for the junction between a DNA duplex and a 5′-single-stranded tail (or flap) and is therefore better described as a 5′-nuclease (2.Lyamichev V. Brow M.A.D. Dahlberg J.E. Science. 1993; 260: 778-783Crossref PubMed Scopus (302) Google Scholar). On a nicked DNA duplex, polymerase-catalyzed primer extension continuously regenerates the substrate for the 5′-nuclease, so that the polymerase effectively drives nick translation (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar). The 5′-nuclease activity of Pol I is present on an independent structural domain encoded by the first one-third of the structural gene, which can be separated from the polymerase by proteolysis or by recombinant DNA manipulations (4.Klenow H. Overgaard-Hansen K. FEBS Lett. 1970; 6: 25-27Crossref PubMed Scopus (54) Google Scholar, 5.Setlow P. Kornberg A. J. Biol. Chem. 1972; 247: 232-240Abstract Full Text PDF PubMed Google Scholar, 6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar). The structure specificity is inherent to the 5′-nuclease domain itself and does not rely on the presence of the polymerase domain (3.Lundquist R. Olivera B. Cell. 1982; 31: 53-60Abstract Full Text PDF PubMed Scopus (57) Google Scholar, 6.Xu Y. Derbyshire V. Ng K. Sun X.C. Grindley N.D.F. Joyce C.M. J. Mol. Biol. 1997; 268: 284-302Crossref PubMed Scopus (50) Google Scholar, 7.Lyamichev V. Brow M.A.D. Varvel V.E. Dahlberg J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6143-6148Crossref PubMed Scopus (63) Google Scholar). Indeed, some bacteriophages encode separate nucleases, having a high degree of homology to the N-terminal region of the bacterial Pol I enzymes, which act in concert with the relevant bacteriophage polymerase (8.Sayers J.R. Eckstein F. J. Biol. Chem. 1990; 265: 18311-18317Abstract Full Text PDF PubMed Google Scholar, 9.Hollingsworth H.C. Nossal N.G. J. Biol. Chem. 1991; 266: 1888-1897Abstract Full Text PDF PubMed Google Scholar, 10.Gutman P.D. Minton K.W. Nucleic Acids Res. 1993; 21: 4406-4407Crossref PubMed Scopus (54) Google Scholar). In eukaryotes and archaebacteria, the structure-specific cleavages required in replication and repair are carried out by "flap endonucleases" (11.Lieber M.R. Bioessays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar, 12.Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 13.Shen B. Qiu J. Hosfield D. Tainer J.A. Trends. Biochem. Sci. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), which exist as independent protein subunits and are therefore able to collaborate with a variety of polymerases. The flap endonuclease family of proteins shows only a modest level of sequence similarity to the bacterial and bacteriophage 5′-nucleases (14.Mueser T.C. Nossal N.G. Hyde C.C. Cell. 1996; 85: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), but the similarity in three-dimensional structures (14.Mueser T.C. Nossal N.G. Hyde C.C. Cell. 1996; 85: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15.Kim Y. Eom S.H. Wang J. Lee D.-S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (328) Google Scholar, 16.Ceska T.A. Sayers J.R. Stier G. Sück D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (164) Google Scholar, 17.Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (147) Google Scholar, 18.Hosfield D.J. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) and in the reactions carried out by these two families of 5′-nucleases leaves little doubt that they are functionally analogous to one another. In vivo, the polymerase and 5′-nuclease of bacterial Pol I must collaborate so as to leave a nick that can be sealed by DNA ligase. Two extreme scenarios can be envisaged. The Pol I molecule might adopt a structure that brings the two active sites into close proximity so they can bind simultaneously to the DNA substrate. Alternatively, the polymerase and 5′-nuclease activities might operate essentially independently of one another, perhaps even with the DNA traveling from one active site to the other via dissociation into free solution. This latter scenario fits well with the existence of a separate 5′-nuclease in some systems and is analogous to the relationship between the polymerase and 3′-5′ exonuclease (editing) functions of Klenow fragment, where the two active sites are separated by about 30 Å, with a substantial amount of the transfer between them occurring via dissociation (19.Joyce C.M. J. Biol. Chem. 1989; 264: 10858-10866Abstract Full Text PDF PubMed Google Scholar). Close juxtaposition of polymerase and 5′-nuclease active sites might be difficult to achieve; the polymerase active site is located at the base of a cleft (20.Brautigam C.A. Steitz T.A. Curr. Opin. Struct. Biol. 1998; 8: 54-63Crossref PubMed Scopus (334) Google Scholar), and the 5′-nuclease domain is structurally complex, possibly requiring threading of the unpaired 5′ end through an arch or loop of the protein (16.Ceska T.A. Sayers J.R. Stier G. Sück D. Nature. 1996; 382: 90-93Crossref PubMed Scopus (164) Google Scholar, 17.Hwang K.Y. Baek K. Kim H.-Y. Cho Y. Nat. Struct. Biol. 1998; 5: 707-713Crossref PubMed Scopus (147) Google Scholar, 18.Hosfield D.J. Mol C.D. Shen B. Tainer J.A. Cell. 1998; 95: 135-146Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). However, recent structures of polymerases with DNA bound at the polymerase site suggest a dislocation of the downstream DNA template beyond the site of synthesis (21.Doublié S. Tabor S. Long A. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1099) Google Scholar, 22.Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar), and such a dislocation might provide a way to accommodate both polymerase and 5′-nuclease active sites in reasonably close proximity. Only two polymerase crystal structures (both of Taq DNA polymerase) include a 5′-nuclease domain. The first structure showed the polymerase and 5′-nuclease sites separated by about 70 Å at opposite ends of a rather extended molecule, arguing in favor of separate and independent active sites; however, x-ray scattering measurements suggested that the polymerase may fold more compactly in solution (15.Kim Y. Eom S.H. Wang J. Lee D.-S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (328) Google Scholar). A more recent crystal structure of Taq DNA polymerase complexed with an Fab showed the nuclease domain (which was remote from the Fab location) close to the fingers subdomain of the polymeras
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