INTRODUCTION

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The small, single-stranded DNA genome of the adeno-associated virus (AAV) is flanked by terminal repeats (TRs), each folding back on itself to form terminal hairpinned structures (Fig. 1). These structures are the only cis elements required for AAV DNA replication, packaging of replicated genomes into capsids, and site-specific integration of provirus into the host genome (15). Each of these distinct activities requires interaction between the AAV nonstructural Rep proteins and the AAV TRs (12, 28, 37).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Model of AAV DNA replication. The boxed region illustrates the steps involved in the terminal resolution of AAV viral ends. In vitro, Rep68 is necessary and sufficient for both the site-specific endonuclease and helicase activities required for terminal resolution. The viral 3' end is indicated with an arrow. Circles depict Rep covalently attached to the viral 5' end at the trs.

The AAV nonstructural proteins, Rep78 and Rep68, contain both ATP-dependent DNA helicase and site-specific endonuclease activities. In our current model of AAV DNA replication, strand-specific nicking at the terminal resolution site (trs) and subsequent Rep-mediated unwinding within the TR generate a 3' primer for the initiation of subsequent DNA repair of the TR, a process referred to as terminal resolution (Fig. 1) (26). During this process, the TR is nicked between the two thymidines in the sequence 3'-GGT/TGA-5', resulting in a 5' phosphotyrosol linkage between Rep and the nicking site (8, 24, 26). Efforts to characterize Rep trs nicking have been facilitated by the development of an in vitro trs endonuclease assay. Site-specific Rep nicking in this reaction is dependent on ATP hydrolysis and the presence of Mg²⁺ (or Mn²⁺) (8, 25, 37). The reason why the Rep transesterification reaction requires ATP hydrolysis has not been clarified.

The ATP dependence of AAV Rep-mediated nicking is a rather novel feature that is shared by the related parvovirus nonstructural protein NS-1 from the mouse minute virus (MVM) (3). However, many other rolling-circle replication initiator proteins do not seem to require ATP for the origin nicking reaction. Notably, the geminivirus Rep protein does not require ATP for in vitro nicking of single-stranded origin substrates. However, this enzyme is unable to nick biologically relevant duplex substrates under these conditions (11). Interestingly, geminivirus replication in vivo is dependent on the formation of a stem-loop structure at the nicking site (20, 27), but the relationship between geminivirus Rep ATPase activity and the formation of this origin stem-loop remains unclear. The Staphylococcus aureus plasmid pT181 also contains an origin stem-loop structure. Extrusion of this stem-loop on supercoiled substrates appears to be dependent on upstream binding of the plasmid-encoded endonuclease RepC, which nicks the origin within the loop region of this structure, but this reaction does not require ATP (9, 17).

Efficient AAV Rep nicking of the hairpinned TR appears to require three sequence recognition elements within the TR. Mutational analysis of the AAV TR has identified a core 22-bp sequence required for stable binding of Rep78 and Rep68 (23). This Rep binding element (RBE) is believed to be the primary recognition element that promotes Rep binding. Interestingly, homologues of this RBE are present at the AAV p5 promoter, in the preferential proviral integration site on human chromosome 19, and within several viral and cellular promoters. Rep78 and Rep68 bind many of these sequences in vitro and appear to regulate AAV transcription through direct interaction with promoter and TR RBEs in vivo (7, 10, 13, 21, 34, 35).

Efficient binding and nicking of hairpinned substrates also require contact between Rep and the small internal palindromes that comprise the tips of the hairpinned TR. The presence of these internal palindromes in hairpinned TRs increases nicking 5- to 50-fold (2, 8, 14, 25). Because the internal palindromes exist in two alternate configurations (flip and flop), it had previously been thought that no specific sequence within this region was recognized. However, Ryan et al. (23) have identified sequence-specific contacts between Rep and a CTTTG motif within the small internal palindromes of the TR. This internal palindrome has a constant position with respect to the trs regardless of the orientation of the TR (flip or flop).

Finally, a specific sequence at the trs itself also appears to be required for nicking. Insertion of a heterologous sequence between the nicking site and the RBE significantly reduces nicking, but nicking occurs at the correct site (25). As yet, however, the specific sequences that comprise the trs have not been identified. Curiously, high-resolution in vitro binding studies do not detect Rep contacts at the trs in the absence of ATP (23). Furthermore, ATP is not an essential cofactor for trs nicking if the trs region is present in a hairpinned TR substrate as single-stranded DNA (25). This observation led us to suggest that ATP-dependent Rep DNA helicase activity is needed to melt the duplex trs to create a single-stranded nicking intermediate. Rep DNA helicase mutants are deficient in trs nicking, supporting this conclusion (31). Additionally, studies of Rep helicase activity have shown that Rep can unwind a duplex DNA substrate provided that it contains an RBE (37).

In our previous work, we identified the sequences within the RBE and the internal palindromes that affect Rep nicking and sequence-specific DNA binding (23). Here we focus on the trs region of the TR. We identify a strand-specific trs core sequence required for Rep-catalyzed nicking through systematic mutation of sequences near the trs. Furthermore, we identify a potential stem-loop structure at the trs. Surprisingly, preferential extrusion of the nicking site stem-loop entirely removes the ATP requirement for Rep nicking. This clearly demonstrates that the AAV Rep protein requires the extrusion of a stem-loop structure containing the nicking site prior to trs cleavage. Furthermore, we conclude that the ATP-dependent Rep DNA helicase activity is necessary to unwind the duplex trs and allow formation of the stem-loop structure prior to nicking.

	MATERIALS AND METHODS

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Purification of baculovirus-expressed Rep68. Rep68 was purified to homogeneity from baculovirus-infected Sf9 cells as previously described (37). Rep68 was purified by sequential chromatography on phenyl-Sepharose, single-stranded DNA-cellulose, and DEAE-cellulose. Preparations were more than 99% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis followed by silver staining (37).

DNA substrates. To construct the synthetic TR substrates used in this study, 100 pmol of two annealed oligonucleotides containing the RBE and the trs sequences was ligated to 500 pmol of a third oligonucleotide containing the secondary structure element. Ligations were done at 32°C in a 100-µl reaction volume containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP, 25 µg of bovine serum albumin per ml, and 1,600 U of T4 DNA ligase (New England Biolabs). Complete TR constructs were purified from ethidium bromide-stained, 10% denaturing polyacrylamide gels containing 50% urea. DNA concentrations of purified substrates were determined by using the Pico Green fluorometric reagent (Molecular Probes). The entire panel of mutants was assayed together to ensure accurate relative DNA concentrations. TR substrates were 5' labeled at 37°C in a 10-µl reaction mixture containing 200 fmol of substrate, 70 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 5 mM dithiothreitol, 20 µCi of [-³²P]ATP, and 20 U of T4 polynucleotide kinase (New England Biolabs). Final concentrations of labeled substrates were determined on the basis of specific activity and confirmed with the Pico Green fluorometric reagent.

trs endonuclease assay. The trs endonuclease reactions were performed essentially as described previously by Im and Muzyczka (8). The 20-µl reaction mixtures contained 5 fmol of 5'-labeled substrate (average, 10⁴ cpm/fmol) and 0.5 mM ATP. The reaction mixtures were incubated at 37°C for 1 h. Proteinase K-digested reaction products were ethanol precipitated, washed with 70% ethanol, and fractionated on 10% denaturing polyacrylamide gels containing 50% urea. The amount of product formed was determined with a phosphorimager (Fuji).

	RESULTS

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Determination of the minimal trs. To determine the minimal trs sequences required for Rep68 nicking, a panel of mutant TR substrates was constructed. These substrates consisted of sequential single-base-pair transversions, beginning 8 bp upstream of the nicking site and extending 7 bp downstream. Mutation of these sequences was facilitated by the use of synthetic oligonucleotides. To construct TR substrates, two annealed oligonucleotides containing the RBE and the trs sequences were ligated to a third oligonucleotide containing the terminal palindromes (Fig. 2A and B). Complete TR constructs were purified from ethidium bromide-stained, denaturing polyacrylamide gels, and DNA concentrations of the purified substrates were determined as described in Materials and Methods. The mutants were then 5'-end-labeled with [-³²P]ATP and T4 polynucleotide kinase, and the final concentrations of labeled substrates were determined on the basis of specific activity and confirmed with fluorometry. Rep trs endonuclease activity on these substrates was assayed in vitro by using homogeneously pure Rep68. All substrates were assayed together, and each substrate was assayed at several Rep concentrations. Typically (Fig. 2C), Rep titrations contained 0.05 to 1 pmol of enzyme in reaction mixtures containing 5 fmol of a given substrate. The results of the nicking reactions with each mutant are shown in Fig. 3.

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Synthetic AAV TR substrates. (A) The sequence of wt AAV TR substrates used in this study is shown. The boxed region denotes the canonical RBE. The positions of the trs and relevant restriction enzyme sites are also indicated. (B) Restriction enzyme analysis was performed on 5'-labeled wt TR substrate. The products were fractionated on a 10% denaturing polyacrylamide gel. TR indicates undigested substrate. (C) Rep68 endonuclease assays were performed on 5'-labeled wt TR substrate (see Materials and Methods for details). Products were resolved on a 10% denaturing polyacrylamide gel. The positions of the 163-nucleotide (nt) substrate and 22-nucleotide product are indicated. The total amount of Rep68 used in the reaction is indicated above each lane.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Rep68 endonuclease assays on 1-bp transversion mutants. Individual base pairs were mutated to transversions and assayed for Rep68 endonuclease activity. Boxed regions denote wt AAV sequence. Arrows point to mutant sequence. Mutants were assayed together as a panel that included wt substrate (see Materials and Methods for details). Rep68 nicking activity on wt substrate is indicated with a horizontal line across the graph. (n = 11 for mutations within the sequence 3'-CCGGTTGAGG-5', and n = 6 for mutations flanking this sequence. Error bars indicate standard deviations of the means.)

Strand specificity of Rep68 trs nicking. Though we had identified a core duplex sequence required for Rep68 nicking, we had not determined the strand specificity of Rep contacts within this sequence. Previous work indicated that insertion of heterologous sequences directly opposite the nick site reduced Rep68 cleavage, yet deletion of these same sequences, resulting in a single-stranded trs, did not reduce nicking. However, the single-stranded trs substrates were nicked at two sites with approximately the same efficiency, the trs and a site 11 nucleotides downstream, indicating a possible role for the opposite strand in Rep68 nicking specificity (25). Hence, the contribution of the strand opposite the trs during Rep68 nicking remained unclear. To clarify these inconsistencies and to determine the strand specificity of Rep68 contacts during nicking, a new panel of trs mutants was constructed. These substrates contained a single-nucleotide transversion either on the trs-containing strand or on the opposite strand (Fig. 4). In this way, each nucleotide of a given base pair was independently mutated within the core trs sequence.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Rep68 endonuclease assays on 1-nucleotide transversion mutants. Individual nucleotides within the 7-bp core sequence were mutated to transversions and assayed for Rep68 endonuclease activity. The sequence mutated in Fig. 3 is shown. Boxed regions denote wt AAV sequence. Arrows point to mutant sequence. Mutants were assayed together as a panel that included wt substrate (see Materials and Methods for details). Relative Rep68 nicking activity on wt substrate is indicated with a horizontal line across the graph. (n = 4 for all mutants. Error bars indicate standard deviations of the means.)

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Rep68 endonuclease reactions on single-stranded TR mutants. (A) The sequence of our substrate designed to mimic the 3' viral end (REANNEAL) is given. The boxed region indicates additional sequences deleted opposite the trs in the extensively single-stranded substrate described by Snyder et al. (25). (B) Rep68 endonuclease reactions were performed on wt and REANNEAL substrates as described in Materials and Methods. Products were resolved on a 10% denaturing polyacrylamide gel. Numbers above lanes indicate total amounts of Rep68 in the reactions expressed in femtomoles.

Secondary structure at the trs. In an attempt to better understand our data, we aligned TR sequences from the seven AAV serotypes. With the exception of AAV5, the region near the trs showed stark conservation among all serotypes. Curiously, this conservation included an inverted repeat flanking the first 5 nucleotides of the trs core sequence (Fig. 6). Additionally, although AAV5 trs sequences were quite disparate with respect to the other serotypes, these sequences also contained an inverted repeat flanking the nick site.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Secondary structure at the AAV trs. (A) Sequences near the trs from the various AAV serotypes were downloaded from GenBank and aligned. Boxed nucleotides denote sequence changes from the consensus. Palindromic sequences are underlined. (B) Palindromic sequences near the AAV2 and AAV5 trs depicted as stem-loop structures. Location of AAV-5 trs is from the work of Chiorini et al. (1). (C) Rep68 nicking data from 1-bp transversion mutants have been superimposed on the predicted AAV2 trs stem-loop structure. Circles denote minimal trs as determined by 1-nucleotide mutants.

View larger version (71K): [in this window] [in a new window]   FIG. 7.   Role of trs stem-loop in Rep68 endonuclease reaction. (A) wt and nostem substrates are depicted after the formation of trs stem-loop structure. The RBE and Rep contacts with the small internal palindromes of the terminal hairpin are indicated with ovals. The minimal trs as determined by 1-nucleotide mutants is also indicated. (B) Rep68 endonuclease reactions were performed on wt and nostem substrates in the presence of 0.5 mM ATP, 0.5 mM ATPS, or no ATP as described in Materials and Methods. Products were resolved on a 10% denaturing polyacrylamide gel. Numbers above lanes indicate the total amount of Rep68 in the reactions expressed in femtomoles. (C) The gel from panel B was phosphorimaged, and the amounts of substrate and product were determined. These data were graphed to show relative specific activities of Rep68 on the two substrates under the different ATP conditions.

Elimination of the ATP requirement for Rep nicking. If extrusion of the trs stem-loop is a necessary step prior to nicking, and if the Rep-associated DNA helicase is necessary for extrusion to occur, then we would expect that the trs nostem substrate (Fig. 7) would be ATP independent for nicking. Indeed, trs endonuclease reactions with the stem-loop mutant were no longer dependent on ATP. Rep cleavage of the mutant was essentially the same in both the presence and the absence of ATP, indicating that ATP was not required for the Rep68 transesterification reaction at the trs (Fig. 7). These data were consistent with Rep68 trs assays using the extensively single-stranded substrate that was nicked in the absence of ATP (25). The single-stranded region of this mutant included the inverted repeat necessary for the predicted trs stem-loop formation (Fig. 5). Hence, it is probable that this partially single-stranded substrate could form the trs stem-loop in solution. However, unlike this single-stranded substrate, the nostem mutant used here was nicked only at the correct site.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Mapping the minimal trs. During the course of this study, we have analyzed the effect of mutations near the AAV trs on Rep68-catalyzed origin nicking. Our results indicate that a 7-base core nucleotide sequence, 3'-CCGGT/TG-5', is required for Rep nicking at the trs. This core sequence is strand specific. It is required only on the nicked strand, and it does not matter if the complementary sequence on the opposite strand is intact. Interestingly, the first 5 nucleotides of this core trs sequence are flanked by an 8-bp inverted repeat that appears to form a stem-loop intermediate prior to nicking. Formation of this stem-loop seems to be ATP dependent and is presumably facilitated by Rep DNA helicase activity acting on the nearby RBE. Conversely, Rep does not require ATP to nick the trs once this stem-loop structure is formed, indicating that the endonuclease reaction is ATP independent.

Architecture of the AAV TR. The finding that specific sequences at the trs are involved in Rep-catalyzed nicking is consistent with our working model of Rep interactions with the AAV TR. In this model, site-specific Rep cleavage at the trs requires sequence-specific interactions with the RBE, the trs, and the small internal palindromes that comprise the tips of the hairpinned TR. In addition to these elements, our data indicate that a stem-loop structure near the trs is also involved in Rep-mediated nicking.

Why is ATP required for Rep68 nicking? Our data indicate that ATP is required during Rep trs nicking to melt the duplex trs and allow formation of a stem-loop structure at the nicking site. Once the trs stem-loop has been formed, the actual Rep nicking reaction does not require ATP. In fact, the Rep68 transesterification reaction appears to be inhibited by bound ATP. Although Rep68 nicking activity on the stem-loop mutant is the same in the absence or in the presence of ATP, the addition of ATPS to the reaction reduced nicking activity to the level observed on wt substrates. Since ATPS is a nonhydrolyzable analogue, it would presumably remain bound to Rep, unlike ATP, which would be hydrolyzed. Thus, it appears that Rep adopts at least two functional conformations during trs nicking, ATP bound and native. The ATP-bound form appears to facilitate Rep helicase activity, and the native form appears to be the more efficient endonuclease.

General mechanisms of origin nicking. The similarity between AAV Rep and other viral replication proteins is not limited to the parvoviruses. Geminivirus origin sequences include a predicted stem-loop structure at the nicking site similar to that in AAV. This structure is required for in vivo replication (20, 27), but the mechanism of stem-loop extrusion is unknown. Although it is tempting to draw mechanistic homologies between AAV Rep and geminivirus Rep, it is worth noting that the geminivirus Rep does not exhibit helicase activity (6). This observation raises questions about the functional significance of the geminivirus Rep ATPase activity. This activity is required for viral DNA replication, but the basis of this requirement is unclear (6). Interestingly, the ATPase activities of both AAV and geminivirus Rep proteins are not DNA dependent (6, 37). This rather novel shared characteristic suggests a functional role for ATP-dependent conformational changes outside the context of DNA unwinding.

Regulation of AAV DNA replication. The extrusion of the AAV trs stem-loop structure appears to be rate-limiting in Rep68 trs nicking. Zhou et al. (37) recently described the reaction kinetics of Rep68 nicking on wt TR substrates as sigmoidal with respect to enzyme concentration. They concluded that at least a dimer was required for Rep68 endonuclease activity on these substrates. However, these studies would not have measured the reaction kinetics of the endonuclease reaction, per se, but would have measured the kinetics of the rate-limiting step of the reaction. Thus, we conclude that the extrusion and stabilization of the stem-loop structure require at least a dimer of Rep68.