pUC19 (2.7 kb [lane 10]) as templates were processed as described in Materials ..... We especially thank Mark Challberg, Charlotte McGuinness, Ken-.
JOURNAL OF VIROLOGY, Apr. 1995, p. 2038–2046 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 69, No. 4
Asymmetric Replication In Vitro from a Human Sequence Element Is Dependent on Adeno-Associated Virus Rep Protein ELENA URCELAY,1 PETER WARD,2 STEPHEN M. WIENER,1 BRIAN SAFER,1 1 AND ROBERT M. KOTIN * Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland, 20892,1 and Department of Microbiology, Cornell University Medical College, New York, New York 100212 Received 11 October 1994/Accepted 3 January 1995
The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3*-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5*-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration. licates only by leading-strand synthesis (for a review, see reference 2). The genome of AAV is single-stranded linear DNA, and either the positive or negative strand is infectious (29). Replication of AAV is dependent on the AAV nonstructural proteins (Rep); either Rep68 or Rep78 is the only AAV protein required for replication in vitro (16, 24). Several activities have been characterized for Rep68 and Rep78 that are involved in viral DNA replication, including binding to the AAV inverted terminal repeat (ITR) (12, 13, 26), sequence-specific DNA binding (6, 42), sequence- and strand-specific endonuclease activity (12), and ATP-dependent DNA helicase activity (12). Second-strand synthesis by cellular replication proteins initiates from the 39-OH of the hairpinned ITR (Fig. 1A) (for reviews of AAV replication, see references 2, 4, and 23). A duplex replication intermediate, which is covalently attached at one end through the ITR (Fig. 1B), requires AAV Rep protein(s) for terminal resolution. Four Rep proteins are produced in cells productively infected with AAV and helper virus: Rep78, Rep68, Rep52, and Rep40. Either Rep68 or Rep78 binds to a specific region within the ITR and nicks one strand of the duplex at a unique site (Fig. 1C). The endonuclease reaction results in covalent attachment of Rep to the newly generated 59 end. The 39 end of the nick serves as a primer for extension of the ITR, possibly by the same type of Pol complex involved in viral second-strand synthesis (Fig. 1D and E). The helicase activity of the covalently attached Rep molecule may unwind the secondary structure of the ITR. The process of terminal resolution provides the means for restoration of the termini and generation of progeny viral genomes (Fig. 1F). Thus, AAV DNA replication can be accounted for entirely by leading-strand synthesis independent of de novo synthesis. Recently, a truncated ITR (DITR) incapable of hairpin formation has been shown to act as a Rep-responsive ori in vitro (6). AAV DNA has the unique property among animal DNA
Mammalian chromosomal DNA replication initiates from sites dispersed throughout the genome with cell cycle regulation. An early step in replication is recognition of origin of replication (ori) sequences by a DNA-binding protein that is thought to function by nucleating polymerase (Pol) complexes for leading- and lagging-strand DNA synthesis (for reviews, see references 3 and 16). Cellular ori sequence elements and cellular factors involved in initiation of replication have not been characterized. Mammalian DNA viruses have been useful as paradigms of cellular DNA replication. These viral ori sequences have been defined (for a review, see reference 5) for simian virus 40 (SV40) (1, 14, 22) and herpes simplex virus (34). Viral proteins required for initiation of replication that function as origin-binding proteins have been characterized, including, e.g., SV40 large-t antigen (T-Ag) (9, 36) and herpes simplex virus UL-9 (25). The development of an in vitro replication system based on SV40 (21, 33, 43) has led to the characterization of cellular proteins necessary for DNA synthesis (for reviews, see references 15 and 32). T-Ag is the only viral gene product necessary for SV40 DNA replication; all other components are cellularly derived. T-Ag hexamers bind to the ori and initiate replication by unwinding the DNA with T-Ag helicase. DNA Pol a-primase complex associates with T-Ag via the B subunit of Pol a (8). Pol d and proliferating cell nuclear antigen displace Pol a-primase and processively extend the leading strand (37–39). Pol a-primase remains associated with T-Ag and catalyzes lagging-strand synthesis. The symmetry of the SV40 ori and T-Ag double hexamer results in bidirectional replication. In contrast, adeno-associated virus type 2 (AAV) DNA rep* Corresponding author. Mailing address: Building 10, Room 7D18, Molecular Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892. Phone: (301) 496-1594. Fax: (301) 496-9985. 2038
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FIG. 1. Replication model for AAV DNA. Thicker lines represent newly synthesized DNA, and the arrowheads indicate directions of synthesis. (A) Input viral DNA; (B) elongation by a cellular replication complex; (C) Rep protein(s) binding to the recognition site within the ITR and nicking one strand of the duplex; (D and E) extension of the ITR by cell replication complex; (F) production of two strands of the viral genome. Each strand consists of newly synthesized and parental DNA. Terminal resolution is represented by steps C, D, and E.
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as described elsewhere (40). The replication reactions were performed as described elsewhere (40), with the following modifications: the total dCTP concentration was increased to 0.1 mM, and the reaction mixtures were preincubated for 1.5 h prior to the addition of [a-32P]dCTP (specific activity, $5,000 Ci/mmol). The latter modification reduces the labeling from the putative repair activities to negligible levels. The concentration of protein in the cell extract was determined by the Bio-Rad colorimetric assay. Each assay was done with 0.1 mg of cellular protein in a final volume of 15 ml that contained 7 mM MgCl2; 4 mM ATP; 200 mM each CTP, GTP, and UTP; 100 mM each dATP, dGTP, and dTTP; 10 mM dCTP; and 10 mCi of [a-32P]dCTP (6,000 Ci/mmol; Amersham), 2 mM dithiothreitol (DTT), 4 mg of bovine serum albumin, 40 mM creatine phosphate (pH 7.7), 2 mg of creatine phosphokinase, 100 mg of HeLa cell extract protein (40), 0.3 mg of $90% supercoiled plasmid. When indicated, 1 mg of the recombinant fusion protein was included (7). Each reaction mixture was incubated at 348C for 18 h. Reactions were terminated by passing over a Sephadex G-50 spin column (59 39, Inc.) and digestion for 2 h at 378C with proteinase K (200 mg/ml) in 7 mM EDTA–0.2% Sodium dodecyl sulfate (SDS)–50 mM NaCl. The nucleic acids were extracted with phenolchloroform, precipitated with ethanol, and dissolved in 2.5 mM Tris-Cl (pH 7.5)–0.25 mM EDTA. Products were fractionated by electrophoresis on 1% (wt/vol) Tris-borate-EDTA (TBE) agarose gels. The gels were dried and exposed to X-ray film (Kodak X-Omat). DpnI and MboI digestions. DNA for DpnI sensitivity assays was prepared as described above, with the exception that no [a-32P]dCTP was added. The DNA samples were digested with increasing amounts of DpnI for 3 h and fractionated on a 1% agarose TBE gel with 0.5 mg of ethidium bromide per ml. The extent of cleavage was determined by UV light-induced fluoresence of the DNA bands in the gel. Sensitivity to MboI was determined using conditions established for complete digestion of amethylated phage lambda DNA. MBP-Rep proteins. The construction of the maltose-binding protein (MBP)Rep expression vectors and production of the MBP-Rep proteins used for in vitro replication reactions have been described in detail elsewhere (6). Briefly, MBP-Rep68D was produced by PCR amplification of the open reading frame (ORF) of rep from codons 3 to 520. The PCR product was cloned in frame with the malE ORF into expression vector pPR997 (New England Biolabs). MBPRep78 was derived from MBP-Rep68D by extension of the amino terminus of the ORF with an overlapping PCR product. MBP-Rep68DNTP contains a substitution of lysine 340 with histidine. The activities of the fusion Rep proteins were determined and were found to be similar to those of wild-type Rep protein (6, 7). Covalent attachment of MBP-Rep78 to the 110-bp P1 element and flanking polylinker sequence. The 110-bp P1 sequence was removed from pMAT50 by EcoRI and HindIII digestion. The fragment was either 59 end labeled with polynucleotide kinase and [g-32P]ATP or 39 end labeled with Klenow fragment
viruses of integration into a defined region of the human genome in chromosome 19 q.13.3 - q.ter (17–19, 30). An explanation to account for this phenomenon has been elusive until the recent finding that Rep bound specifically to a defined region (P1) within the integration locus that has a sequence element similar to that of the Rep-binding sequence in the AAV ITR (see Fig. 2) (42). Because of these newly described findings and to extend the model for targeted integration, we tested the cloned chromosome 19 Rep-binding element (pMAT50) in an in vitro replication assay. MATERIALS AND METHODS Plasmids. A SmaI subfragment derived from the human chromosome 19 integration site for AAV DNA (see Fig. 2) (18, 19) has previously been shown to bind Rep68 and Rep78 was inserted into the SmaI site of pUC19 (42). Cloned full-length, wild-type AAV DNA has been described elsewhere (20). Plasmid pSVoriAAV was produced by isolating a 326-bp AvrII-PvuII fragment corresponding to nucleotides (nt) 5187 to 270 of SV40 DNA. The overhangs of the restriction sites were blunted with T4 DNA polymerase and inserted into the EcoRV site of pBluescript SK(1). The 4.4-kb MscI fragment of pAV2 was inserted into the SmaI site of the plasmid containing the SV40 sequences. Plasmid pHisD is an 11,000-bp plasmid that contains the bacterial gene for histidinol dehydrogenase (HisD) and a portion of the genomic sequence for the mouse hypoxanthine phosphoribosyltransferase gene (28). In vitro replication reactions. Cell extracts were prepared from HeLa S3 cells
FIG. 2. Sequence alignment of the P1 element derived from the chromosome 19 integration locus (AAVS1) with the AAV ITR. AAVS1 is represented schematically by the positions of the CpG island (filled box) (16), and sites of proviral integration are indicated (hatched region) (16, 28, 29). P1 was obtained as a SmaI subfragment of AAVS1 (nt 354 to 468 [16]; see also reference 42). The Rep-binding motifs of both P1 and AAV ITR are outlined and labeled. The sequences homologous to the TRS of AAV are outlined in both the ITR sequence and P1. The endonuclease cleavage site is between the thymidines within the TRS box and is indicated by the arrow.
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FIG. 3. Rep requirement in trans for replication. Template requirement for stimulation of [32P]dCMP incorporation into replication products. Both functional Rep protein and a sequence element that functions as a Rep-dependent ori are necessary for specific radiolabeling of the template. (A) Replication assay mixtures containing 0.3 mg of either supercoiled pAV2 (9.0 kb [lanes 1, 2, and 3]), pMAT50 (2.8 kb [lanes 4, 5, and 6]), pHisD (11.0 kb [lanes 7, 8, and 9]), or pUC19 (2.7 kb [lane 10]) as templates were processed as described in Materials and Methods. Rep proteins were included as indicated. N, MBP-Rep68DNTP; 68, MBP-Rep68D; 78, MBP-Rep78. Size markers are in kilobases (M). The open-circular (O) or linear (L) forms of pMAT50 are indicated. (B) Replication assay mixtures containing 0.3 mg of pSVoriAAV (7.4 kb [lanes 1, 2, and 3]) or pAV2 (9.0 kb [lanes 4, 5, and 6]) were processed as described in Materials and Methods. The linear (L) and rescued (R) products of pAV2 are indicated. The 4.7-kb rescued product is derived from the AAV moiety of pAV2, and the 4.2-kb product corresponds to the vector moiety.
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FIG. 4. (A) Extent of DNA synthesis determined by DpnI resistance. Replication reactions were performed as described in Materials and Methods by using either pMAT50 or pHisD as the template. Replication reactions that included MBP-Rep68D (68) or MBP-Rep78 (78) are as indicated. Samples were digested with 60 U of DpnI (New England Biolabs) in 0.21 M NaCl for 12 h at 378C or 10 U of MboI (New England Biolabs) for 3 h in reaction buffer supplied by the manufacturer. Digested samples are indicated by 1, no enzyme treatment is indicated by 2. Lane M, 1-kb ladder. Sizes are indicated in kilobases. The appropriate enzyme concentrations were determined by analyzing the digestion products with increasing amounts of enzyme after ethidium bromide agarose gel electrophoresis (data not shown). pMAT50 processed from replication reactions without [a-32P]dCTP was used as the substrate for standardization of DpnI digestion. The substrate for MboI standardization was bacteriophage lambda DNA produced in dam mutant E. coli (data not shown). (B) Circular map of pMAT50 with the locations of DpnI-MboI sites indicated by numbers alone. The recognition sites for the following endonucleases are shown: AlwNI, BglI, HindIII, EcoRI, SphI, and SspI. The P1 element is represented by the filled thick line flanked by SmaI sites (nt 412 to 520). The positions of b-lactamase gene (Ap), plasmid ori, and lacZ ORF are labeled.
and [a-32P]dCTP. Following polyacrylamide gel electrophoresis (PAGE) purification, the substrates were digested with either PstI or KpnI. Digestion with PstI resulted in EcoRI end-labeled products, while KpnI digestion yielded HindIII end-labeled probes. The end-labeled probes were purified by phenol-chloroform extraction and ethanol precipitation. MBP-Rep78 (1.4 mg) was incubated with the probes in a final volume of 25 ml of core buffer (12 mM Tris-Cl [pH 7.4], 40 mM NaCl, 1 mM EDTA, 0.1% Triton X-100 [wt/vol], 1 mM dithiothreitol, 4% glycerol [wt/vol]; 2.5 mM ATP and 25 mM MgCl2 were added as indicated). The probes were added ('15,000 cpm) and incubated for 30 min at 308C. The reaction was stopped with an equal volume of 23 SDS gel loading buffer. The products of this reaction were run on a 10% denaturing SDS gel to detect covalent bond formation between protein and P1 probe. Synthetic oligonucleotides. Oligonucleotides containing 57 bp of P1 sequence
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FIG. 5. Replication using linearized plasmids as substrates. pMAT50 was linearized prior to incubation with MBP-Rep68D. Following extraction of protein, the DNA was digested with the restriction enzymes indicated. (A) pMAT50 linearized with SspI. Lanes: 1, postreplication digestion with BglI (B); 2, no postreplication digestion; 3, postreplication digestion with SphI (S); M, 1-kb size markers (Gibco-BRL) that were 39 end labeled. A diagram of SspI-linearized pMAT50 is shown underneath the autoradiograph, with the relative positions of the BglI and SphI sites indicated. The 110-bp P1 element is represented by the filled rectangle. SspI and BglI digestion of pMAT50 generated three fragments, respectively, of 1,678, 688, and 430 bp. The 430-bp fragment remained unlabeled. SspI and SphI digestion produced two fragments, respectively, of 2,058 and 738 bp. Both fragments were labeled. (B) pMAT50 linearized with AlwNI prior to the replication reaction. Lanes: 1, no postreplication digestion; 2, postreplication digestion with EcoRI (E); 3, postreplication digestion with HinDIII (H); M, 1-kb size markers (Gibco-BRL) that were 39 end labeled. The diagram beneath the autoradiograph indicates the relative positions of EcoRI-, HindIII-, and AlwNI-cut sites. The position of the P1 element is represented by the filled rectangle. (C) MBP-Rep68D requirement for the replication of pMAT50. The substrate, pMAT50, was linearized prior to the replication reaction with either EcoRI (lanes 1 and 2) or NdeI (lanes 3 and 4). MBP-Rep68DNTP was included in the reaction mixtures labeled N; MBP-Rep68D was included in the lanes labeled R. Lane M, 1-kb size markers (Gibco-BRL) that were 39 end labeled.
were synthesized and PAGE purified (Midland Certified Reagant Company). The P1 sequence contained both the Rep-binding motif (GCTC)3 and the 5-bp sequence homologous to the AAV terminal resolution site (TRS) site. Three oligonucleotides were utilized for covalent attachment and endonuclease activity reactions. NP-1 is complementary to NP-3. NP-2 is a 39-terminal deletion of NP-3. The sequences are as follows: NP-1, 59-TACGTCCCGCCCGCCCAGCGAGC GAGCGAGCGCCGAGCCCCAACCGCCGCCACCAGTCATG; NP-2, 59-CA TGACTGGTGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGG GCGGGCGGGA; NP-3, 59-CATGACTGGTGGCGGCGGTTGGGGCTCGG CGCTCGCTCGCTCGCTGGGCGGGCGGGACGTA. The duplex oligonucleotide NP-1–NP-2 was uniquely 39 end labeled by filling in a 4-base 59 overhang with Klenow and [a-32P]dCTP. Unincorporated deoxynucleoside triphosphates were removed by a G-50 Sephadex spin column, and the oligonucleotides were purified by phenol-chloroform extraction and ethanol precipitation. A total of 120,000 cpm of probe (specific activity, 2.2 3 107 cpm/mg) per reaction was used. NP-3 (1 mg) was 59 end labeled with [g-32P]ATP (3,000 Ci/mmol) and T4 polynucleotide kinase (New England BioLabs) using the reaction buffer that was supplied. Unincorporated ATP was removed by a G-50 Sephadex spin column. Covalent attachment of MBP-Rep78 to synthetic oligonucleotides. The covalent attachment of MBP-Rep78 to the oligonucleotides was performed as described elsewhere (21), except that the reaction was carried out at 378C for 10 min. The amounts of MBP-Rep78 ranged from 0.028 to 0.14 mg. Endonuclease cleavage site determination. The endonuclease cleavage site was mapped by methods previously described (6). 32P-59-end-labeled oligonucleotide NP-3 was annealed with unlabeled NP-1. A 20-ng amount of duplex oligonucleotides was used in each reaction. The reaction mixtures were incubated for 30 min at 378C. The reactions were terminated and deproteinated by the addition of 40 ml of stop buffer (10 mM Tris-Cl [pH 7.9], 10 mM NaCl, 0.5% SDS, 0.2 mg of yeast tRNA per ml, 20 mM EDTA, 2 mg of proteinase K per ml). The reaction mixtures were incubated for 30 min at 378C. The nucleic acids were extracted with phenol-chloroform and ethanol precipated. The dried samples were resuspended in gel loading buffer (80% formamide, 0.025% bromophenol blue, 0.025% xylene cyanole FF) and were denatured by heating to 808C. The samples were fractionated electrophoretically on an 8% polyacrylamide–8 M urea sequencing gel.
RESULTS Rep68 and HeLa cellular factors catalyze P1-dependent DNA replication. The 110-bp human chromosome 19 element, P1 (Fig. 2), binds specifically to wild-type Rep68 (42) and to recombinant Rep protein produced as a fusion protein in Escherichia coli (MBP-Rep78 or MBP-Rep68D) (6). To test whether binding of Rep to human DNA may facilitate integration of AAV DNA into human chromosome 19, the P1 element cloned into pUC19, pMAT50 (42), was used as a template in an in vitro replication assay. Cellular extract served as the source of DNA polymerases and other replication factors (40). Template pAV2 served as a positive control for Repspecific replication and rescue (6, 41). Using template containing the P1 element and either MBP-Rep68D or MBP-Rep78, two radiolabeled bands comigrating with linear and open circular forms of pMAT50 were detected (Fig. 3A, lanes 5 and 6). The differences in the amounts of radiolabel incorporated in reactions with either MBP-Rep68D or MBP-Rep78 (Fig. 3) varied among different reactions and between different extracts and may not reflect distinctions in activity between the two recombinant proteins. In contrast, MBP-Rep68DNTP resulted in relatively little incorporation of [32P]dCMP (Fig. 3A, lanes 1, 4, and 7). The nucleoside triphosphate (NTP)-binding mutant of MBP-Rep68D has been previously characterized and shown to bind to the ITR and DITR but not to have helicase or endonuclease activities associated with wild-type Rep (6). The activities of MBP-Rep68DNTP were found to be similar to those of the NTP mutant of Rep68, K340H (26, 27). In the
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FIG. 6. (A) Labeled-strand specificity determined by hybridization to singlestranded DNA. Approximately 100 ng of DNA was applied to each slot and adsorbed to a Zeta-Probe membrane (Bio-Rad). Each strip contains three slots: duplex pBluescript SK (2) (pBst), single-stranded pBluescript SK (1), and (2) DNA. 32P-59-end labeled oligonucleotides specific to the T3 or T7 promoters were used as probes to confirm strand specificity. T3, 59-ATTAACCCTCACTA AAGGGA; T7, 59-TAATACGACTCACTATAGGG (strips 1 and 2, respectively). Open-circular DNA radiolabeled in the replication reaction was agarose gel purified, electroeluted, and used as a hybridization probe. Probes derived from MBP-68D and MBP-Rep78 were used for hybridizations of strips 3 and 4. E. coli DH11S (Gibco-BRL) transformed with either pBluescript SK (1) or (2) were infected with helper phage M13KO7 to produce single-stranded pBluescript. The hybridization medium consisted of 7% SDS, 0.1 M NaPi (pH 7.4), 1 mM EDTA, and 0.1 mg of denatured salmon sperm DNA per ml. The high-stringency wash solution was 0.23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate)– 0.1% SDS. The T3 and T7 probes were hybridized at 428C, and the strips were washed at room temperature. The MBP-Rep68D and MBP-Rep78 probes were hybridized at 658C, and the strips were washed at 658C. (B) Measurement of radioactivity associated with the samples shown in panel A. Activities were determined by phosphoimaging, and the results are graphically displayed.
presence of either MBP-Rep68D or MBP-Rep78 and a plasmid containing wild-type AAV DNA, both rescued and circular forms of pAV2 were detected (Fig. 3A, lanes 2 and 3). The requirement for Rep and either the AAV ITR or the P1 element for specific replication is in contrast to results obtained using a DNA sequence lacking the P1 element or AAV ori (pHisD) (28). Very little incorporation was detected in reactions with pHisD as a template and in which either MBPRep68DNTP, MBP-Rep68D, or MBP-Rep78 was added (Fig. 3A, lanes 7, 8, and 9, respectively). The labeling of pMAT50 was dependent on the addition of MBP-Rep68D or MBPRep78 to the reaction. Either pUC19 (Fig. 3A, lane 10) or
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pBluescript (data not shown) (7) was unable to serve as a template. As an additional control for Rep effects on known oris, plasmid pSVoriAAV was used as a template for the in vitro replication reaction (6). This plasmid consists of the SV40 ori cloned into a plasmid containing an AAV genome lacking both ITRs. Plasmid pAV2 consists of the wild-type AAV genome. Little or no specific incorporation of [32P]dCMP was detected with pSVoriAAV template reactions (Fig. 3B, lanes 1, 2, and 3). The presence of two viral ITRs in pAV2 resulted in radiolabeled linear and rescued products as previously described (11, 40, 41). These results indicate that Rep-dependent replication is likely to involve DNA recognition by Rep. Further support of specificity is provided by the negative results obtained with a larger plasmid (pHisD) (Fig. 3A, lanes 7, 8, and 9) or a heterologous origin of replication derived from SV40 DNA (pSVoriAAV) (Fig. 3B, lanes 1, 2, and 3). Estimating the extent of radiolabel incorporated into pAV2 and pMAT50 indicates that the P1 element serves almost as efficiently as the two viral oris in pAV2 (Fig. 3A, lanes 2 and 5 and lanes 3 and 6). This is consistent with a previous report that demonstrated that a plasmid containing the AD9 portion of the AAV ITR (Fig. 2) stimulated incorporation of [32P]dCMP into acid-precipitable material with similar efficiency as pAV2 (6). That is, using the levels of P-32 incorporation as an indicator of DNA synthesis, a nonhairpinned ori element is utilized with results similar to those with a hairpinned ori. pMAT50 replication products are DpnI and MboI resistant. The extent of DNA synthesis can be established by resistance to DpnI endonuclease. This has much greater activity when both adenosines in the recognition sequence are methylated. Such methylation is not performed by eukaryotic methylases. Therefore, conversion to DpnI resistance indicates that hemimethylation or lack of methylation is a result of one or two rounds of DNA replication in a eukaryotic host, respectively, whereas the restriction endonuclease MboI will cleave DNA only when both adenosines in the enzyme recognition site are unmethylated. Thus, two rounds of replication will yield radiolabeled products that are DpnI resistant and MboI sensitive. However, one round of replication generates radiolabeled products that are both DpnI and MboI resistant as a result of hemimethylation of the duplex DNA. The results indicate that a band of mobility corresponding to the open circular form ('3.2 kb) of pMAT50 is resistant to either MboI (Fig. 4A, lanes 2 and 3) or DpnI (Fig. 4A, lanes 8 and 9). Linear forms of pMAT50 appear to be produced as a consequence of digestion with either endonuclease. The locations of DpnI-MboI recognition sites are unsymmetrically distributed around pUC19, and no sites are within the P1 element inserted into the unique Sma site (Fig. 4B). The simplest explanation for the formation of linear products after DpnI digestion is that DNA synthesis proceeds at least 80% around the circular template and terminates prior to completion of the full-length product. The DpnI site at position 277 would then be bimethylated and sensitive to cleavage. Similarly, the minor amount of linear-size product present in the MboI-treated samples could be explained by inefficient second-round synthesis terminating within 1 kb of initiation. The open circular form of the 3.6-kb band was confirmed by restriction endonuclease digestion of the isolated product. pMAT50 replication is asymmetric. To determine the directionality of pMAT50 replication, linearized templates were utilized. The template pMAT50 was digested to completion with either SspI (Fig. 5A) or AlwNI (Fig. 5B) (shown diagrammatically in Fig. 4B). Following the replication reaction, the SspI-linearized DNA was digested with BglI or SphI, and the AlwNI-linearized template was digested with HindIII or EcoRI
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FIG. 7. Covalent linkage activity of MBP Rep protein to P1. (A) Diagram of key features of P1 sequence. The Rep-binding motif (GCTC)3 and the region homologous to the AAV TRS are indicated. (B) Radiolabeled P1 probes. Probes were prepared as follows. The EcoRI-to-HindIII fragment, which includes the P1 element, was radiolabeled with 32P on either strand at the HindIII site (59 HindIII [59 D3] or 39 HindIII [39 D3]). The probes were incubated in core buffer (27) with either MBP-Rep78 or MBP-Rep68DNTP. ATP and MgCl2 were included when indicated. The positions of free probe and probe covalently attached to MBP-Rep78 are indicated. The products were fractionated by electrophoresis on a 10% polyacrylamide gel containing SDS. (C) Effect of increasing protein concentration on covalent bond formation between MBP-Rep78 and P1 oligonucleotide. Synthetic complementary oligonucleotides containing 57 bp of P1 sequence were uniquely 39 end labeled and examined for their ability to form a covalent bond with MBP-Rep78 as described elsewhere (see reference 8 and Materials and Methods). The products were fractionated on a 4 to 20% gradient polyacrylamide gel containing SDS (Bio-Rad). The positions of the free probe P1 and the covalent MBP-Rep78:P1 complex are indicated. Lanes: 1, no MBPRep78; 2, empty lane; 3 to 9, increasing concentrations of MBP-Rep78 as follows: 0.0068 (lane 3), 0.0136 (lane 4), 0.028 (lane 5), 0.056 (lane 6), 0.0840 (lane 7), 0.112 (lane 8), and 0.140 (lane 9) mg. Molecular mass standards are in kilodaltons. (D) No added Rep control. Each end of the fragment diagrammed in panel A was uniquely end labeled at the positions indicated. RI, EcoRI site; D3, HindIII site. The reaction mixtures were treated with or without proteinase K as indicated (0.4% SDS–0.74 mg of proteinase K per ml at 558C for 20 min). The reaction mixtures were incubated for 30 min at 378C and fractionated on a 10% polyacrylamide gel containing 0.1% SDS. The positions of the free probes
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(Fig. 5). The pattern of labeled fragments from each set can best be explained by predominantly unidirectional replication originating from within the P1 element and proceeding to the 59 end of the template strand. The BglI-digested products of the SspI-linearized template resulted in radiolabeling two of the three fragments. The unlabeled fragment of 430 bp is located proximally to the SspI site. Digestion with SphI generated two fragments, of 2,058 and 738 bp, and both fragments were labeled. The lack of incorporated label into the 430-bp SspI-BglI fragment, together with the labeled 738-bp SspI-SphI fragment, indicates that replication most likely originates from the region between the BglI and SphI sites. Results consistent with these data were obtained with AlwNI-linearized substrate (Fig. 5B). The replication products were digested with either HindIII or EcoRI, which have unique sites flanking the P1 insert. The predominantly labeled smaller fragment indicates a strong bias in replication direction. The relatively low amount of label incorporated into the larger fragment may be the result of nonspecific or repair activity. The specificity of linearized DNA replication for MBPRep68D is demonstrated by the inability to use MBPRep68DNTP (Fig. 5C) or MBP-Lacz (data not shown). These proteins provide a control for the presence of copurified bacterial proteins which might have activity on the substrates in the reaction. These results imply that binding per se of MBPRep68D or MBP-Rep78 to a functional ori is not sufficient for replication. Thus, the incorporation of [a-32P]dCMP into plasmid-size DNA is dependent on the presence of the P1 sequence in cis and MBP-Rep68D in trans. Corroborating evidence for asymmetrical replication is provided by the use of the labeled open-circular replication products as probes against single-stranded DNA derived from rescued pBluescript SK(1) or (2). The hybridization results shown in Fig. 6 indicate that there is preferential labeling of one strand of DNA. The T3 and T7 probes demonstrate the specificity of the target DNA (Fig. 6A, lanes 1 and 2). The radiolabeled DNA derived from the MBP-Rep68D- and MBPRep78-containing reaction mixtures both hybridized preferentially to the plus strand of pBluescript. PhosphorImager analysis of the gel established that the hybridization signal to the plus strand is approximately 2.5-fold greater than the signal to the minus strand (Fig. 6B). The observed bias in the direction of replication is likely to be a low estimate, since labeling of pMAT50 by nonspecific repair activities contributes to the hybridization signal of both the plus and the minus strands. Labeling of minus strands of pMAT50 is as predicted from the results obtained with the SspI- or AlwNI-linearized pMAT50 templates. Rep68 binding is accompanied by site-specific endonuclease activity and 5*-covalent attachment. The mechanism of terminal resolution of the AAV ITR requires that Rep cleave one strand of duplex DNA at a defined site within the ITR that is a fixed distance from the Rep-binding motif. If replication of pMAT50 is initiated by a similar mechanism, then formation of a covalent intermediate of MBP-Rep68D–DNA would be predicted. Such a protein-DNA complex would be stable upon SDS-PAGE and would be distinct from free probe or nonco-
are shown. Positions of molecular mass standards are on the right in kilodaltons. Following electrophoresis, the gel was fixed, dried, and exposed for autoradiography. (E) Experiment similar to that described in panel D, except that 0.8 mg of MBP-Rep78 was included in each reaction. Following incubation, half of the reaction mixture was treated with proteinase K when indicated. The mobilities of the free probes and complexed probes are indicated. Molecular mass standards are in kilodaltons.
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URCELAY ET AL.
J. VIROL.
FIG. 8. Rep endonuclease cleavage of P1. 32P-59-end-labeled oligonucleotide NP-3 was annealed with unlabeled NP-1 (see Materials and Methods). Approximately 20 ng of duplex oligonucleotides was used as a substrate in each 20-ml reaction mixture. Following incubation, the reaction mixtures were processed as described and fractionated on an 8% sequencing gel. Following electrophoresis, the gel was fixed, dried, and exposed to X-ray film for autoradiography. The positions of full-length substrate and cleavage product are indicated by the arrows labeled NP-3 and product. The sequence of the oligonucleotide is on the left, with the Rep-binding motif and TRS homolog bracketed. (A) Approximately 0.7 mg of the indicated protein was added. Lanes: 1 and 5, purine-specific sequencing reaction; 2, MBP-Rep78; 3, MBP-Rep68DNTP; 4, no added protein. (B) Dose response of endonuclease activity. Increasing amounts of MBP-Rep78 correspond to amounts of product generated. The reactions were performed as described for panel A. Lanes: 1 and 9; purine-specific sequencing reaction; 2, no added protein; 3 to 8, 0.09, 0.18, 0.4, 0.7, 1.4, and 2.8 mg, respectively.
valent protein-DNA complexes. The cloned P1 element was excised from pUC19 and uniquely end labeled for use as a substrate in the TRS endonuclease assay (Fig. 7A). The lowermobility band consists of 39-end-labeled DNA covalently linked to protein according to a previous characterization (7) (Fig. 7B). Complementary oligonucleotides that corresponded to the P1 sequence were synthesized (see Materials and Methods) and used to determine whether the TRS endonuclease activity was responsive to increasing MBP-Rep78 concentrations. The extent of covalent protein-DNA production was proportional to the the concentration of MBP-Rep78 in the system (Fig. 7C). The cleavage site may correspond to a 5- out of 6-nt match of the TRS of the AAV ITR. The reaction mixtures were treated with proteinase K to ascertain whether the lower-mobility bands that were identified as complex (Fig. 7B and C) were composed of proteinDNA. The strand-specific complex that formed with the 39end-labeled HindIII site was sensitive to the protease treatment (Fig. 7E [sample 39D3]), whereas proteinase treatments of reactions with no added Rep were unaffected (Fig. 7D [sample 39D3]). These results are consistent with previous data that established that covalent linkage of Rep to DNA occurs as a consequence of endonuclease activity (see below) (7, 13). Site-specific endonuclease activity of MBP-Rep78 on P1. If Rep initiated cellular DNA synthesis by a mechanism analogous to that of AAV DNA replication, then a critical early step
in this process is nicking a single strand of DNA at a defined site. Previous studies have demonstrated that MBP-Rep proteins could bind to linear duplex substrates with affinities similar to those of hairpinned ITR substrates (7). However, the endonuclease activity of Rep was 50- to 100-fold less efficient on the nonhairpinned substrates (6). To test whether MBPRep could nick at a site within the P1 element, oligonucleotide NP-3 was 59 end labeled, annealed to unlabeled NP-1, and incubated with MBP-Rep78, MBP-Rep68DNTP (Fig. 8A, lanes 2 and 3), or MBP-Rep68D (data not shown). The position of the full-length NP-3 is indicated. A purine-specific sequencing ladder (Fig. 8A, lanes 1 and 5) allows precise mapping of the cleavage products. The most prominent band produced by incubation with MBP-Rep78 is indicated by the arrow labeled product. A fragment of this size could be produced by cleavage of NP-3 within the TRS homolog motif and corresponds to cleavage predominantly between the two thymidines. A fragment of this size is not observed either with the NTP mutant or with no added protein (Fig. 8A, lanes 3 and 4). Similar reactions were performed with increasing amounts of MBP-Rep78 (Fig. 8B). The intensity of the product band is proportional to the amount of MBP-Rep78 in the reaction. Other cleavage products become more pronounced also as the concentration of Rep increases. This imprecision may be an inherent property of Rep or may be due to the nature of the Rep protein that is employed, i.e., Rep that is bacterially expressed as a fusion protein.
VOL. 69, 1995
HUMAN DNA REPLICATION INITIATED BY AAV Rep PROTEIN
FIG. 9. Comparison of AAV terminal resolution with a model of Rep-dependent plasmid replication. (A to E) Terminal resolution of the AAV genome; (A9 to E9) Rep-dependent replication from a non-ITR origin, e.g., P1; (A and A9) Rep binding to its recognition sequence adjacent to a properly positioned endonuclease site (vertical arrow); (B and B9) single-stranded cleavage at the endonuclease site and covalent attachment of Rep to the 59 end of the nick; (C and C9) assembled Pol complex (closed and open circles) extending the nontemplate strand from the 39-OH of the nick; (D) extension of the ITR by DNA leading-strand synthesis; (D9) extension of the nonviral origin by leading-strand DNA synthesis.
DISCUSSION The identification of a Rep-responsive, human ori provides the basis for a model of DNA leading-strand synthesis. A process involving unidirectional replication neatly dissects two complex and interrelated replication reactions, i.e., leadingand lagging-strand DNA synthesis. The model for Rep68- or Rep78-dependent replication of DNA containing the P1 (or DITR) origin is derived from the known in vitro activities of the Rep proteins (Fig. 9). These include sequence-specific binding (6, 7, 42), strand- and site-specific endonuclease activity (12), helicase activity (12), and stimulation of replication in vitro from an AAV ori (6, 24). The Rep-binding motif has been determined and occurs in both the AAV ITR and P1 (Fig. 2) (6, 42). A dissociation constant of '10210 M has been determined for MBP-Rep68D binding to hairpin wild-type ITR or linear D57ITR (7). DNA synthesis initiates from the 39-OH generated by the endonuclease activity of Rep at the TRS within P1 independently of RNA priming. This is analogous to AAV DNA replication. The asymmetry of the TRS with respect to the Rep-binding motif constrains elongation from a single site on one strand, i.e., unidirectional. This model predicts displacement of the nontemplate strand as the replication complex proceeds (Fig. 9D9). These replication intermediates were not detected, presumably because of the sensitivity of single-stranded DNA to nucleases in the cell extract. The cleavage at the TRS involves the formation of a stable Rep-thymidine:39-DNA intermediate (13). The ATP-dependent helicase activity of Rep68 and Rep78 (12) may obviate the need for cellular helicases to promote unwinding of the DNA for elongation. The covalently attached Rep molecule is then released by an undetermined mechanism. The nucleoprotein intermediate is stable, as demonstrated by in vitro assays (13), although Rep has not been detected in the mature AAV virion.
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The model of AAV Rep protein-mediated replication of pMAT50 appears analogous to rolling-circle replication models described for Staphylococcus aureus plasmid pT181 (35) or bacteriophage fX174 (10). The parallel with staphylococcal pT181 replication is so striking that the components of the models are interchangeable. The staphylococcal RepD protein has origin specific-binding activity and single-strand, site-specific endonuclease activity that results in covalent attachment of RepD to the 59 end of the nick via a phosphotyrosine linkage. Unidirectional replication initiates from the free 39-OH and proceeds around the plasmid. The staphylococcal RepD protein may remain covalently attached to the 59 end of the displaced strand throughout replication. The similarity to the bacterial system provides a precedent for AAV Rep68- or Rep78-dependent replication of a circular template. The many similarities between the two systems suggest independent convergent evolution of this mode of replication. Alternatively, evolutionary conservation of protein functions could account for the similarities observed. Targeted integration of AAV DNA into human chromosome 19. AAV DNA integrates into a small, defined region of human chromosome 19 in cultured cells at a frequency of approximately 70% (19, 30). The integration junctions are distinct at the molecular level among the latently infected cell lines analyzed. Comparison of AAV DNA sequence with AAVS1 sequence confirmed that integration occurred by nonhomologous recombination (17, 30). The interaction of Rep with either chromosome 19-derived DNA (42) or AAV ITR (6, 12, 13, 26) strengthens the argument for Rep involvement in targeted integration. The data presented here indicate that Rep is required and allow a model to be formulated for targeted integration. The process of AAV DNA integration now appears to be the result of limited DNA synthesis initiated by binding of Rep to the P1 element within AAVS1. The recombination mechanism may involve subunit exchange between Rep complexes associated with each substrate. The Rep-binding motif has been found within other genes (GenBank release 80). However, the requirement for a properly positioned TRS would decrease the probability of occurrence in random sequence to #6 3 10211, thereby defining a unique sequence. A recombination model involving limited DNA synthesis is supported by previous descriptions of proviral structures (19, 30, 31). For some latently infected cell lines, duplication of cellular sequences adjacent to the provirus has been described. Significantly, all of the characterized integration events were asymmetrically distributed with respect to the P1 element. This recombination model may constitute a new recombination pathway that utilizes functions intrinsic to the cell. In vitro reconstitution of DNA synthesis using cloned P1 or DITR as template, MBP-Rep, and purified cellular proteins may be possible. The large quantities of MBP-Rep available provide an opportunity to identify cellular components involved in leading-strand synthesis without the additional complication of lagging-strand synthesis. The involvement of Rep in specialized cellular DNA synthesis provides the basis for a targeted integration model of AAV DNA integration into human chromosome 19. ACKNOWLEDGMENTS We especially thank Mark Challberg, Charlotte McGuinness, Kenneth Berns, Bernard Moss, and Nancy Nossal for helpful discussions and comments. We are grateful to Jay Chiorini, Roland Owens, and Sirrka Kyo ¨stio ¨ for critical reading and suggestions that were useful in the preparation of the manuscript. This work was supported in part by NHLBI CRADA 91-02 with Genetic Therapy, Inc., Gaithersburg, Md.
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1. Bergsma, D. J., D. M. Olive, S. W. Hartzell, and K. N. Subramanian. 1982. Territorial limits and functional anatomy of the simian virus 40 replication origin. Proc. Natl. Acad. Sci. USA 79:381–385. 2. Berns, K. I. 1990. Parvovirus replication. Microbiol. Rev. 54:316–329. 3. Brewer, B. J. 1994. Intergenic DNA and the sequence requirements for replication initiation in eukaryotes. Curr. Opin. Genet. Dev. 4:196–202. 4. Carter, B. J. 1992. Adeno-associated virus vectors. Curr. Opin. Biotechnol. 3:533–539. 5. Challberg, M. D., and T. J. Kelly. 1989. Animal virus DNA replication. Annu. Rev. Biochem. 58:671–717. 6. Chiorini, J. A., M. D. Weitzman, R. A. Owens, E. Urcelay, B. Safer, and R. M. Kotin. 1994. Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli. J. Virol. 68:797–804. 7. Chiorini, J. A., S. M. Wiener, S. R. M. Kyo¨stio¨, R. A. Owens, R. M. Kotin, and B. Safer. 1994. Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats. J. Virol. 68:7448–7457. 8. Collins, K. L., A. A. Russo, B. Y. Tseng, and T. J. Kelly. 1993. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication. EMBO J. 12:4555–4566. 9. DeLucia, A. L., B. A. Lewton, R. Tjian, and P. Tegtmeyer. 1983. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication. J. Virol. 46:143–150. 10. Eisenberg, E., J. F. Scott, and A. Kornberg. 1976. An enzyme system for replication of duplex circular DNA: the replicative form of phage fX174. Proc. Natl. Acad. Sci. USA 73:1594–1597. 11. Hong, G., P. Ward, and K. I. Berns. 1992. In vitro replication of adenoassociated virus DNA. Proc. Natl. Acad. Sci. USA 89:4673–4677. 12. Im, D.-S., and N. Muzyczka. 1990. The AAV origin binding protein is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61:447–457. 13. Im, D.-S., and N. Muzyczka. 1989. Factors that bind to adeno-associated virus terminal repeats. J. Virol. 63:3095–3104. 14. Jones, K. A., and R. Tjian. 1984. Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference. Cell 36:155–162. 15. Kelly, T. J. 1991. DNA replication in mammalian cells: insights from the SV40 model system. Harvey Lect. 85:173–188. 16. Kelman, Z., and M. O’Donnell. 1994. DNA replication: enzymology and mechanisms. Curr. Opin. Genet. Dev. 4:185–195. 17. Kotin, R. M., R. M. Linden, and K. I. Berns. 1992. Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 11:5071–5078. 18. Kotin, R. M., J. C. Menninger, D. C. Ward, and K. I. Berns. 1991. Mapping and direct visualization of a region-specific viral DNA integration site on chromosome 19q13-qter. Genomics 10:831–834. 19. Kotin, R. M., M. Siniscalco, R. J. Samulski, X. Zhu, L. Hunter, C. A. Laughlin, S. McLaughlin, N. Muzyczka, M. Rocchi, and K. I. Berns. 1990. Site-specific integration by adeno-associated virus. Proc. Natl. Acad. Sci. USA 87:2211–2215. 20. Laughlin, C. A., J. D. Tratschin, H. Coon, and B. J. Carter. 1983. Cloning of infectious adeno-associated virus genomes in bacterial plasmids. Gene 23: 65–73. 21. Li, J. J., and T. J. Kelly. 1985. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol. Cell. Biol. 5:1238–1246. 22. Li, J. J., K. W. Peden, R. A. Dixon, and T. Kelly. 1986. Functional organization of the simian virus 40 origin of DNA replication. Mol. Cell. Biol. 6:1117–1128. 23. Muzyczka, N. 1992. Use of adeno-associated virus as a general transduction
J. VIROL. vector for mammalian cells. Curr. Top. Microbiol. Immunol. 158:97–129. 24. Ni, T.-H., X. Zhou, D. M. McCarty, I. Zolotukhin, and N. Muzyczka. 1994. In vitro replication of adeno-associated virus DNA. J. Virol. 68:1128–1138. 25. Olivo, P. D., N. J. Nelson, and M. D. Challberg. 1988. Herpes simplex virus DNA replication: the UL9 gene encodes an origin binding protein. Proc. Natl. Acad. Sci. USA 85:5414–5418. 26. Owens, R. A., J. P. Trempe, N. Chejanovsky, and B. J. Carter. 1991. Adenoassociated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin. Virology 184:14–22. 27. Owens, R. A., M. D. Weitzman, S. R. Kyo ¨stio ¨, and B. J. Carter. 1993. Identification of a DNA-binding domain in the amino terminus of adenoassociated virus Rep proteins. J. Virol. 67:997–1005. 28. Reid, L. H., E. G. Shesley, H.-S. Kim, and O. Smithies. 1991. Cotransformation and gene targeting in mouse embryonic stem cells. Mol. Cell. Biol. 11:2769–2777. 29. Samulski, R. J., L. S. Chang, and T. Shenk. 1987. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J. Virol. 61:3096–3101. 30. Samulski, R. J., X. Zhu, X. Xiao, J. D. Brook, D. E. Housman, N. Epstein, and L. A. Hunter. 1991. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 10:3941–3950. (Erratum, 11:1228, 1992.) 31. Shelling, A. N., and M. G. Smith. 1994. Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene. Gene Ther. 2:1–5. 32. Stillman, B., S. P. Bell, A. Dutta, and Y. Marahrens. 1992. DNA replication and the cell cycle. CIBA Found. Symp. 170:147–156. 33. Stillman, B. W., and Y. Gluzman. 1985. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol. Cell. Biol. 5:2051–2060. 34. Stow, N. D. 1982. Localization of an origin of replication within the TRs/IRs repeated region of the herpes simplex virus type 1 genome. EMBO J. 1:863– 867. 35. Thomas, C. D., D. F. Balson, and W. V. Shaw. 1990. In vitro studies of the iniation of Staphylococcal plasmid replication. J. Biol. Chem. 265:5519–5530. 36. Tjian, R. 1978. The binding site of SV40 DNA for a T-antigen related protein. Cell 13:165–179. 37. Tsurimoto, T., and B. Stillman. 1991. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primertemplate junction by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem. 266:1950–1960. 38. Tsurimoto, T., and B. Stillman. 1991. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J. Biol. Chem. 266:1961–1968. 39. Waga, S., and B. Stillman. 1994. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature (London) 369:207–212. 40. Ward, P., and K. I. Berns. 1991. In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome. J. Mol. Biol. 218:791–804. 41. Ward, P., E. Urcelay, R. Kotin, B. Safer, and K. Berns. 1994. Adenoassociated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein. J. Virol. 68:6029–6037. 42. Weitzman, M. D., S. R. M. Kyo¨stio ¨, R. M. Kotin, and R. A. Owens. 1994. Adeno-associated virus (AAV) rep proteins mediate complex formation between AAV DNA and the human integration site. Proc. Natl. Acad. Sci. USA 91:5808–5817. 43. Wobbe, C. R., F. B. Dean, Y. Murakami, J. A. Borowiec, P. Bullock, and J. Hurwitz. 1987. In vitro replication of DNA containing either the SV40 or the polyoma origin. Philos. Trans. R. Soc. Lond. B Biol. Sci. 317:439–453.