Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP·trinucleotide complex (pTP·CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP·pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP·C and pTP·CA were still bound to the polymerase, after the formation of pTP·CAT 60% of the pTP·pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and inK m for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA … instead of the natural GTAGTA … ) we show that G5 rather than G3 is used to start, leading to a pTP·tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two. Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP·trinucleotide complex (pTP·CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP·pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP·C and pTP·CA were still bound to the polymerase, after the formation of pTP·CAT 60% of the pTP·pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and inK m for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA … instead of the natural GTAGTA … ) we show that G5 rather than G3 is used to start, leading to a pTP·tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two. The replication of the linear 36-kilobase pair adenovirus (Ad) 1The abbreviations used are: Ad, adenovirus; pol, polymerase; pTP, precursor of terminal protein; dd, dideoxy.1The abbreviations used are: Ad, adenovirus; pol, polymerase; pTP, precursor of terminal protein; dd, dideoxy. DNA initiates at two origins located at either molecular end employing a protein-priming mechanism. Initiation requires at least two viral proteins, the DNA polymerase (pol) and the precursor of the terminal protein (pTP), which acts as the primer and is linked covalently to the first nucleotide, a dCMP molecule. Initiation can be stimulated by the cellular transcription factors NFI and Oct-1 which guide the pTP·pol complex to the core origin employing their DNA binding domains. This leads to stabilization of the preinitiation complex and increases the number of initiation events by enhancing the V max of the reaction. In addition, the virus-coded single-stranded DNA-binding protein stimulates initiation by lowering the K m for dCTP (1Mul Y.M. van der Vliet P.C. Nucleic Acids Res. 1993; 21: 641-647Crossref PubMed Scopus (33) Google Scholar). The virus uses a jumping back mechanism for initiation. At position 4 from the 3′-end of the template a pTP·CAT intermediate is synthesized which jumps back to be paired to the template residues 1–3 before elongation starts (2King A. van der Vliet P.C. EMBO J. 1994; 13: 5786-5792Crossref PubMed Scopus (72) Google Scholar). This jumping or sliding-back mechanism was first described for φ29 (3Méndez J. Blanco L. Esteban J.A. Bernad A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9579-9583Crossref PubMed Scopus (98) Google Scholar) and appears to be universal for protein-priming replication systems (4Caldentey J. Blanco L. Bamford D.H. Salas M. Nucleic Acids Res. 1993; 21: 3725-3730Crossref PubMed Scopus (67) Google Scholar, 5Martı́n A.C. Blanco L. Garcı́a P. Salas M. Méndez J. J. Mol. Biol. 1996; 260: 369-377Crossref PubMed Scopus (33) Google Scholar, 6Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 131-176Google Scholar), enabling errors made during initiation and small deletions to be restored. Subsequent elongation concomitant with the displacement of the non-template strand further requires the Ad DNA-binding protein in addition to the polymerase (for review, see Refs. 7Hay R.T. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 699-720Google Scholar and 8van der Vliet P.C. Curr. Top. Microbiol. Immunol. 1995; 199: 1-30PubMed Google Scholar). The multiprotein preinitiation complex assembled at the origin consists of five proteins that interact with DNA and with each other with different affinities. Several of the protein-protein interactions have been studied individually. The polymerase interacts with the NFI DNA binding domain (9Mermod N. O'Neill E.A. Kelly T. Tijan R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (536) Google Scholar, 10Armentero M.T. Horwitz M. Mermod N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11537-11541Crossref PubMed Scopus (39) Google Scholar, 11Dekker J. Van Oosterhout J.A.W.M. van der Vliet P.C. Mol. Cell. Biol. 1996; 16: 4073-4080Crossref PubMed Scopus (27) Google Scholar) and possibly also with the DNA-binding protein. pTP contacts the Oct-1 POU homeodomain (12Coenjaerts F.E.J. Van Oosterhout J.A.W.M. van der Vliet P.C. EMBO J. 1994; 13: 5401-5409Crossref PubMed Scopus (44) Google Scholar, 13van Leeuwen H.C. Rensen M. van der Vliet P.C. J. Biol. Chem. 1997; 272: 3398-3405Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These interactions are mostly weak, and dissociation occurs already at limited (200 mm) salt concentrations. In contrast, pTP and pol form a stable heterodimer that can only be dissociated in vitrounder strong denaturing conditions (1.7 m urea) (14Lichy J.H. Field J. Horwitz M.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5225-5229Crossref PubMed Scopus (53) Google Scholar) or by binding of specific antibodies (15Webster A. Leith I.R. Hay R.T. J. Virol. 1994; 68: 7292-7300Crossref PubMed Google Scholar, 16Webster A. Leith I.R. Hay R.T. J. Virol. 1997; 71: 539-547Crossref PubMed Google Scholar). We are interested in the dynamic aspects of association and dissociation events occurring during initiation as well as during the transition to chain elongation. Previous studies employing an immobilized DNA replication system have shown that the interaction between NFI and pTP·pol is disrupted already early in initiation (17Coenjaerts F.E.J. van der Vliet P.C. Nucleic Acids Res. 1994; 22: 5235-5240Crossref PubMed Scopus (10) Google Scholar), in agreement with its role as temporarily acting recruitment factor. Recently dissociation of Oct-1 from pTP·pol complex was shown to take place upon elongation (13van Leeuwen H.C. Rensen M. van der Vliet P.C. J. Biol. Chem. 1997; 272: 3398-3405Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). No information exists about dissociation of the pTP·pol complex. A priori these two proteins could stay together during replication, but this would require high flexibility of the end of the nascent strand anchored to the pTP which would have to be present in the moving fork. We have studied the dissociation of pTP and polymerase employing separation of initiation and elongation products by glycerol gradient centrifugation and find that the heterodimer is disrupted early after the synthesis of the pTP·CAT initiation intermediate. This is accompanied by changes in the catalytic properties of the polymerase. Moreover, we provide evidence for flexibility in the choice of the position in the origin at which the formation of initiation intermediates starts. Ad serotype 5 TP-DNA was isolated as described previously (18Coenjaerts F.E.J. van der Vliet P.C. Methods Enzymol. 1995; 262: 548-560Crossref PubMed Scopus (10) Google Scholar). The [α-32P]dNTPs were from ICN, and [γ-32P]ATP (3,000 Ci/mmol) was obtained from Amersham International. Unlabeled deoxynucleotides, dideoxynucleotides, and oligonucleotide templates were obtained from Pharmacia Biotech Inc. The sequence of the wild-type 30-mer containing the template strand of the Ad5 origin is 3′-GTAGTAGTTATTATATGGAATAAAACCTAA-5′. The template strand of the GA repeat template is 3′-GAGAGAGATATTATATGGAATAAAACCTAA-5′. Oligonucleotides SP1 (5′-GATCACAGTGAGTAC) and SP1C+6 (5′-TCTATTGTACTCACTGTGATC) were purified by electrophoresis on 8m urea, 20% polyacrylamide gel electrophoresis. Oligonucleotide SP1 was 5′-end labeled with [γ-32P]ATP and T4 polynucleotide kinase and purified further by polyacrylamide gel electrophoresis. Partially double-stranded primer-template structures were made by hybridizing labeled SP1 to the nonlabeled SP1C+6 oligonucleotide in the presence of 0.2 m NaCl and 60 mm Tris-HCl, pH 7.5. The mixture was heated at 70 °C and was allowed to cool down slowly to room temperature. The pTP·pol complex and the free Ad DNA polymerase were expressed in Sf-9 cells employing recombinant baculoviruses that were constructed as described Ref. 19Watson C.J. Hay R.T. Nucleic Acids Res. 1990; 18: 1167-1173Crossref PubMed Scopus (16) Google Scholar. Purification of both the pTP·pol complex and the free Ad DNA polymerase to apparent homogeneity was described previously (20King A.J. Teertstra W.R. Blanco L. Salas M. van der Vliet P.C. Nucleic Acids Res. 1997; 25: 1745-1752Crossref PubMed Scopus (24) Google Scholar). Polyclonal antibodies were raised against the pTP·pol complex in rabbits as described before (12Coenjaerts F.E.J. Van Oosterhout J.A.W.M. van der Vliet P.C. EMBO J. 1994; 13: 5401-5409Crossref PubMed Scopus (44) Google Scholar). The standard incubation mixture (25 μl) for initiation contained 0.4 μg of pTP·pol and 0.7 μg of single-stranded oligonucleotide as template in a buffer containing 20 mm Hepes, pH 7.5, 1 mmdithiothreitol, 1 mm MgCl2, 1 μg of bovine serum albumin, and NaCl to a final concentration of 55 mm. The initiation reaction was allowed to proceed for 1 h unless otherwise indicated at 30 °C in the presence of a 0.75 μm concentration of one of the [α-32P]dNTPs (5 μCi). The reaction was stopped by the addition of 10 μl of 0.25 m sodium pyrophosphate and 2 μl of 0.2 m EDTA. Reaction products were precipitated with 5 μl of trichloroacetic acid (20% final concentration) for 20–30 min at 0 °C. The precipitate was spun down by centrifugation for 15 min at 12,000 rpm in an Eppendorf centrifuge, and the pellet was dissolved in Laemmli buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 100 mm Tris, pH 9.0, 0.02% were heated for 5 min at 100 °C and separated by electrophoresis in and by Initiation to elongation was conditions to for initiation, for the of the four was labeled with (5 0.75 μm unless otherwise The other were and present at was by 20 μm This in an elongation after position the first in the the studies the of the were as indicated in the were as described for the initiation reaction. The standard incubation mixture (200 μl) for glycerol gradient contained μg of pTP·pol and 5 μg of single-stranded oligonucleotide or template 4 μg of Ad5 or 5 μg of double-stranded oligonucleotide in a buffer containing 20 mm Hepes, pH 7.5, 1 mm 1 mm MgCl2, μg of bovine serum albumin, and NaCl to a final concentration of 55 mm. Replication was allowed to proceed for 60 min at 30 °C in the presence of 0.75 μm and and incubation a was on a glycerol gradient as described and a was for were on of a linear glycerol gradient containing mm Hepes, pH 7.5, 1 mmdithiothreitol, 1 mm mm and were for h at rpm in an at 4 °C. of μl were and by electrophoresis in replication products were by or The pTP·pol complex was by an as described before (12Coenjaerts F.E.J. Van Oosterhout J.A.W.M. van der Vliet P.C. EMBO J. 1994; 13: 5401-5409Crossref PubMed Scopus (44) Google Scholar). with 5 and as molecular were of μl were and of the 5 μl of the was with μl of mm μl of 5 in the presence of mm Tris, pH and 100 mm after an incubation of 10 min the in that is in this reaction was by the at the position of in the 10 μl of the was with were a was from The between SP1 with SP1C+6 containing a end of nucleotides was used as a primer-template for DNA incubation of μl contained of the of the Ad DNA polymerase in a buffer containing pH 7.5, 1 mm glycerol, bovine serum albumin, and a of the four a for 15 min at 20 were by the addition of a final concentration of 1 mm. incubation at °C for the indicated the were stopped by the addition of to 10 mm. were by 20% polyacrylamide gel electrophoresis in m and We a template containing the single-stranded Ad5 origin with pTP·pol and a concentration of μm μm and 20 μm these conditions elongation position the first in the can take also of the pTP·CAT At dCTP pTP·CAT products can be that is an intermediate in replication (1Mul Y.M. van der Vliet P.C. Nucleic Acids Res. 1993; 21: 641-647Crossref PubMed Scopus (33) Google Scholar). dCTP of the intermediate to the of both products in one reaction. The reaction products were subsequently separated by glycerol gradient the interaction between proteins and DNA we at in the This concentration was that the pTP·pol heterodimer as as during or high salt were we dissociation of the pTP·pol by were and by with a raised against the pTP·pol which on pTP and is against 1 A that of the pTP with the polymerase the of the which to the position of the pTP·pol present in The replication the position of the protein and with its molecular that the DNA is double-stranded of This that this replication dissociated from the polymerase. In contrast, the pTP·CAT intermediate was two one with and one at the pTP·pol that of the pTP·CAT is still bound to the polymerase. Employing a single-stranded origin that was at position from to we also the of a This to the Replication products on the natural TP-DNA template and in the a dissociation of the replication products, with dissociated and the pTP·CAT intermediate two A and The were obtained at various pTP·pol or template or NFI was in the reaction the of dissociation in more we the reaction in the presence of one or two and nucleotides only the formation of pTP·C or pTP·CA In to both products with the pTP·pol complex This that dissociation only starts after the synthesis of pTP·CAT formation the intermediate jumps back. is that jumping back and dissociation are this we used an oligonucleotide template the first three nucleotides and at position … this template jumping back is and the presence of a at position the use of the first in the template for pTP·CAT replication on this template in the presence of and in the synthesis of pTP·CAT only Subsequent in a glycerol gradient a of pTP·CAT to that with the of a These that dissociation occurs of the jumping back Dissociation of the pTP·pol complex that the free polymerase elongation. the of the free and enzyme we purified Ad DNA polymerase and pTP·pol complex from recombinant cells and the of DNA synthesis on a of the free and the polymerase were with four nucleotides and a primer paired to a template in the of initiation of the reaction by the addition of 1 mm MgCl2, of the primer was allowed for the indicated and the products were on a sequence shown in of were 30 by DNA polymerase, this only after min for the pTP·pol complex. of the products we a of elongation for the free polymerase with the pTP·pol that dissociation from pTP after initiation the DNA polymerase more efficient for elongation. we that the properties of free polymerase and dissociated DNA polymerase are but is to the properties of the dissociated polymerase only a small than of pTP·pol in the reaction. we that the conditions for formation of from during elongation. The initiation reaction is to and strong inhibitors of elongation van der Vliet P.C. Nucleic Acids Res. PubMed Scopus (24) Google Scholar, T. J.H. Hurwitz J. Proc. Natl. Acad. U. S. A. PubMed Scopus Google Scholar, J.H. Horwitz M.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, van der Vliet P.C. Curr. Top. Microbiol. Immunol. Scholar, Y.M. van R.T. van der Vliet P.C. Nucleic Acids Res. 1989; PubMed Scopus Google Scholar). Moreover, the K m for dCTP is during initiation (1Mul Y.M. van der Vliet P.C. Nucleic Acids Res. 1993; 21: 641-647Crossref PubMed Scopus (33) Google Scholar). were obtained we m for and the formation of the pTP·CAT of and μm were for this was and The various are in The for these is that changes in the polymerase active site upon transition from initiation to concomitant with dissociation from pTP and jumping of the various properties of the Ad DNA polymerase during initiation and DNA-binding protein; μm Y.M. van der Vliet P.C. Nucleic Acids Res. 1993; 21: 641-647Crossref PubMed Scopus (33) Google μm Y.M. van R.T. van der Vliet P.C. Nucleic Acids Res. 1989; PubMed Scopus Google at at van der Vliet P.C. Nucleic Acids Res. PubMed Scopus (24) Google to 100 at T. J.H. Hurwitz J. Proc. Natl. Acad. U. S. A. PubMed Scopus Google Scholar, J.H. Horwitz M.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google to at m for dCTP were in the presence and of Ad which the K m for both initiation and elongation. DNA-binding protein; in a K m for dCTP were in the presence and of Ad which the K m for both initiation and elongation. the Ad5 template … ) the is used for the synthesis of the pTP·CAT intermediate. whether is the repeat that is used by the Ad5 DNA polymerase, we a template with a terminal repeat of four GA … which occurs in on the Ad5 template (2King A. van der Vliet P.C. EMBO J. 1994; 13: 5786-5792Crossref PubMed Scopus (72) Google Scholar), only dCMP could be of the was position was on the GA repeat as with wild-type 5 which of the 4 residues was used for initiation we the A residues at the and one by one to a and studied of nucleotides in the intermediate elongation shown in the initiation intermediate could only be labeled with and employing the and and that the synthesis of the intermediate had from template residues 1 or and the intermediate could also be labeled with and These are with a start of synthesis from template G5 from which a pTP·tetranucleotide is synthesized which jumps back to elongation. the and these intermediates would be and the could be by the synthesis of a mixture of and from the use of both G5 and which would also in with and T. We the by 20 μm elongation on the a would have been at G5 this would have been to back and elongation. in the of synthesis of the presence of the at the 3′-end would and this is we In for Ad5 DNA polymerase the choice of the to be by the repeat but rather by the position of this A preference exists for position 4 or 5 rather than that a repeat is still present and starts with a This indicates a of flexibility in the choice of the the properties of four well replication products and we have obtained strong evidence for dissociation of the pTP primer and the DNA polymerase early during the initiation In these was to the proteins from the template the pTP·pol interaction We the interaction between pTP and pol is to be stable (14Lichy J.H. Field J. Horwitz M.S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5225-5229Crossref PubMed Scopus (53) Google Scholar), the of in with high salt or high disrupted the complex. We conditions of which the pTP·pol complex the contacts with DNA were We dissociation of the pTP·pol complex after the synthesis of the pTP·CAT intermediate. 60% of the pTP·CAT becomes dissociated. dissociation is that the events dissociation take place of the events occurring after the pTP·CAT formation is the jumping back a between dissociation and jumping back could be established the replication by employing the template which jumping back of pTP·CAT is the as the wild-type elongation of the pTP·CAT intermediate dissociation occurs and is nucleotides have been We have strong that other jumping are place after pTP·CAT Before and after pTP·CAT formation the catalytic properties of the DNA polymerase as by changes in the K m for and and by changes in in This indicates a change in the active site of Ad DNA polymerase after pTP·CAT a conformational change in the polymerase active site also the pTP interaction domain of the polymerase and dissociation of the pTP·pol complex. in DNA polymerase the dissociation of two was accompanied by a conformational change J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). a role was for or pTP in upon studies employing isolated and antibodies against the these antibodies were well and the also in cells Van van der Vliet P.C. PubMed Scopus Google Scholar). we a role for pTP in for to its to to the J.A. J. Virol. 1993; PubMed Google Scholar), a role in vitro that a of DNA replication the virus a dissociation of pTP from the polymerase is a the polymerase is more efficient after the release of pTP and Moreover, dissociation of pTP the enabling (20King A.J. Teertstra W.R. Blanco L. Salas M. van der Vliet P.C. Nucleic Acids Res. 1997; 25: 1745-1752Crossref PubMed Scopus (24) Google Scholar). We have previously the initiation site used in Ad5 the template sequence … ) at position from which a pTP·trinucleotide intermediate is templates containing a different sequence of a of two nucleotides … ) and position G5 is and a pTP·tetranucleotide intermediate is The of the initiation site is by the template sequence rather than by the from the molecular end or the pTP·pol binding that the binding of pTP·pol to the core origin is of the the molecular this a of flexibility in a of the template in the active of a complex of the of DNA polymerase and the complex of DNA polymerase show that DNA can a the flexibility in contacts J. 1996; PubMed Scopus Google Scholar). a of the first to pTP is a suggesting that this is an of Ad5 for a specific are in other protein-priming DNA replication systems that use a initiation mechanism (3Méndez J. Blanco L. Esteban J.A. Bernad A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9579-9583Crossref PubMed Scopus (98) Google Scholar, J. Blanco L. Bamford D.H. Salas M. Nucleic Acids Res. 1993; 21: 3725-3730Crossref PubMed Scopus (67) Google Scholar, 5Martı́n A.C. Blanco L. Garcı́a P. Salas M. Méndez J. J. Mol. Biol. 1996; 260: 369-377Crossref PubMed Scopus (33) Google Scholar). φ29 and to have a to start replication from the and template of the in this position (3Méndez J. Blanco L. Esteban J.A. Bernad A. Salas M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9579-9583Crossref PubMed Scopus (98) Google Scholar, 5Martı́n A.C. Blanco L. Garcı́a P. Salas M. Méndez J. J. Mol. Biol. 1996; 260: 369-377Crossref PubMed Scopus (33) Google Scholar), that the position rather than the the start In the of a limited of flexibility in the position to be by the for a in the Employing the GA repeat the Ad5 DNA polymerase to start from position G5 rather than the that on the natural template the start is at would G5 be terminal present in adenovirus M. A. T. PubMed Scopus (27) Google Scholar) the formation of a pTP·trinucleotide or The of an initiation intermediate of three or four nucleotides with could be to this requires the synthesis of at least three or four nucleotides to at least in pol Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We are to R. Hay and for the of Ad DNA polymerase and Ad