Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.     

Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen. The active site, located at the bottom of a positively charged cleft, is formed by the spatial convergence of a divalent metal ion and two conserved sequence motifs that define the rolling circle replication superfamily.     

Interaction of the adeno-associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat.

We have characterized a Rep binding sequence which is within the A stem region of the adeno-associated virus terminal repeat (TR) and compared its affinity with that of the complete hairpinned TR for pure Rep68. Both the A stem and the complete TR substrates produced a complex pattern of protein-DNA complexes in which at least six different bound species could be distinguished. Competition experiments suggested that the dissociation constant for the A stem sequence is approximately 125-fold higher than that for the complete TR. The competition experiments also suggested that the average number of Rep molecules per TR substrate molecule under conditions of saturating substrate is 3.7:1, while for the A stem substrate, the ratio is 10:1. In spite of the apparent difference in protein-to-DNA ratio in the complexes, no major difference was seen in the mobility or the pattern of the protein-DNA complexes with the two kinds of substrates, suggesting that the difference in protein-to-DNA ratio was due to the lower stability of the A stem complex rather than the actual number of Rep molecules per DNA molecule. At least some of the difference in stability of the two kinds of complexes was due to the fact that the dissociation rate of the A stem substrate from the protein-DNA complexes was approximately fourfold faster than that of the complete TR. The dissociation rate curves for both substrates, however, were complex, suggesting that substrate was being released from at least two different kinds of protein-DNA complexes at different rates. In addition, we have analyzed binding to several substitution mutants within the A stem of the TR. A five-base mutant near the terminal resolution site (trs site) had little effect on binding. Two other mutants produced seven- or five-base substitutions within the 25-bp sequence of the A stem that had been identified in the accompanying report (D. M. McCarty, D. J. Pereira, I. Zolotukhin, X. Zhou, J. H. Ryan, and N. Muzyczka, J. Virol. 68:4988-4997, 1994) as essential for binding. Each of these mutants eliminated some but not all of the repeating GAGC motifs in the 25-bp A stem region. Both of these mutants completely abolished binding to the A stem substrate but only partially reduced binding in the context of the complete hairpinned TR. Furthermore, neither mutant altered the pattern of Rep-DNA complexes produced.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.     

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.     

Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb

Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.     

Determination of adeno-associated virus Rep68 and Rep78 binding sites by random sequence oligonucleotide selection.

To further define the canonical binding site for the P5-promoted Rep proteins of the adeno-associated virus, a modified random oligonucleotide selection procedure was performed, using purified recombinant Rep protein. These results may explain the effects of Rep on cellular gene expression.     

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations. Activation of ATPase activity of Rep68 by DNA is cooperative with a Hill coefficient of 1.8 +/- 0.2. When examined by gel filtration at 0.5 M NaCl in the absence of DNA, Rep68 self-associates in a concentration-dependent manner. In the presence of DNA containing the binding element, Rep68 (and Rep78) forms protein-DNA complexes that exhibit concentration-dependent self-association in gel filtration analysis. The ATPase activity of the isolated Rep68-DNA and Rep78-DNA complexes is not activated by additional target DNA. Results of sedimentation velocity experiments in the presence of saturating target DNA are consistent with Rep68 forming a hexamer of the protein with two copies of the DNA element. Activation of the ATPase activity of Rep68 is associated with the formation of a protein-DNA oligomer.     

Roles of adeno-associated virus Rep protein and human chromosome 19 in site-specific recombination

Adeno-associated virus type 2 (AAV) is the only known eucaryotic virus capable of targeted integration in human cells. AAV integrates preferentially into human chromosome (ch) 19q13.3qter. The nonstructural proteins of AAV-2, Rep78 and Rep68, are essential for targeted integration. Rep78 and Rep68 are multifunctional proteins with diverse biochemical activities, including site-specific binding to AAV and ch-19 target sequences, helicase activity, and strand-specific, site-specific endonuclease activities. Both a Rep DNA binding element (RBE) and a nicking site essential for AAV replication present within the viral terminal repeats are also located on ch-19. Recently, identical RBE sequences have been identified at other locations in the human genome. This fact raises numerous questions concerning AAV targeted integration; specifically, how many RBE sequences are in the human genome? How does Rep discriminate between these and the ch-19 RBE sequence? Does Rep interact with all sites and, if so, how is targeted integration within a fixed time frame facilitated? To better characterize the role of Rep in targeted integration, we established a Rep-dependent filter DNA binding assay using a highly purified Rep-68 fusion protein. Electron microscopy (EM) analysis was also performed to determine the characteristics of the Rep-RBE interaction. Our results determined that the Rep affinity for ch-19 is not distinct compared to other RBEs in the human genome when utilizing naked DNA. In fact, a minimum-binding site (GAGYGAGC) efficiently associated with Rep, suggesting that as many as 2 × 10⁵ sites may exist. In addition, such sites also exist frequently in nonprimate mammalian genomes, although AAV integrates site specifically into primate genomes. EM analysis demonstrated that only one Rep-DNA complex was formed on ch-19 target DNA. Surprisingly, identically sized complexes were observed on all substrates containing a RBE sequence, but never on DNA lacking an RBE. Rep-DNA complexes involved a multimeric protein structure that spanned ca. 60 bp. Immunoprecipitation of AAV latently infected cells determined that 1,000 to 4,000 copies of Rep78 and Rep68 protein are expressed per cell. Comparison of the Rep association constant with those of established DNA binding proteins indicates that sufficient molecules of Rep are present to interact with all potential RBE sites. Moreover, Rep expression in the absence of AAV cis-acting substrate resulted in Rep-dependent amplification and rearrangement of the target sequence in ch-19. This result suggests that this locus is a hot spot for Rep-dependent recombination. Finally, we engineered mice to carry a single 2.7-kb human ch-19 insertion containing the AAV ch-19 target locus. Using cells derived from these mice, we demonstrated that this sequence was sufficient for site-specific recombination after infection with transducing vectors expressing Rep. This result indicates that any host factors required for targeting are conserved between human and mouse. Furthermore, the human ch-19 cis sequences and chromatin structure required for site-specific recombination are contained within this fragment. Overall, these results indicate that the specificity of targeted recombination to human ch-19 is not dictated by differential Rep affinities for RBE sites. Instead, specificity is likely dictated by human ch-19 sequences that serve as a Rep protein-mediated origin of replication, thus facilitating viral targeting through Rep-Rep interactions and host enzymes, resulting in site-specific recombination. Control of specificity is clearly dictated by the ch-19 sequences, since transfer of these sequences into the mouse genome are sufficient to achieve Rep-dependent site-specific integration.Adeno-associated virus type 2 (AAV) contains a single-stranded DNA genome of approximately 4.7 kb (50) and is a member of the Parvoviridae family (3). AAV is unique among other eucaryotic DNA viruses in that it utilizes a biphasic lifecycle to persist in nature. In the presence of a helper virus, adenovirus (Ad) or herpesvirus, AAV will undergo a productive infection. In the absence of a helper virus, AAV will integrate preferentially (>70%) into chromosome (ch) 19q13.3qter (3, 35). The ability of this nonpathogenic DNA virus, or virus-derived vector systems, to integrate site specifically have made it an attractive candidate vector for human gene therapy (45).The AAV genome consists of two open reading frames (ORFs), which comprise the rep and cap genes, and 145-bp inverted terminal repeats (ITRs), which serve as the origins of replication (3, 35). The left ORF of AAV encodes four nonstructural proteins, Rep78, Rep68, Rep52, and Rep40. Extensive characterization of Rep78 and Rep68 in vitro has identified the following biochemical activities, DNA binding (18, 19), site-specific and strand-specific endonuclease activities (17, 19), and DNA-RNA and DNA-DNA helicase activities (17, 19, 59), all of which appear to be necessary for viral replication (15, 53). More importantly, Rep78 and Rep68 are required for mediating targeted integration (2, 43, 47, 51, 60).Though site-specific integration is dependent upon either of the two large Rep proteins, the AAV ITRs are the only cis elements required for integration (34, 44, 61). In the absence of Rep proteins, the virus will still integrate through the ITR sequence but randomly into the host genome (21, 56, 61). Although integration in the absence of the Rep proteins is random, virus-cell junctions are nearly identical to junctions formed during targeted integration (DNA microhomology at junctions, specific deletions of the ITR sequences, rearrangement of the chromosome locus, and head-to-tail virus concatemers) (41, 62). In fact, in vitro integration products generated using cellular extracts produced identical type junctions, demonstrating the essential role the ITRs play in viral integration (62). From this analysis, Yang et al. (62) concluded that both random and targeted integration are dependent upon a cellular recombination pathway, with the role of Rep facilitating integration at ch-19. To help account for AAV targeting, a nearly identical Rep binding element (RBE) and a nicking site (trs) to that present on the AAV ITR was identified on the ch19.13.3qter AAV integration sequence (23–25, 43, 46, 54, 57). It was also demonstrated that Rep68 could mediate complex formation between the AAV ITR and the ch-19 integration site in vitro (57). This led to a hypothesis that AAV may target integration by Rep-mediated complex formation between the AAV ITR and the ch19 integration site. However, since this observation subsequent data has demonstrated that Rep can bind to degenerate RBE sequences, (5, 32). In fact, computer analysis identified at least 15 genomic genes which contained RBE sites that bound to AAV Rep protein in vitro, all more efficient than the ch-19 sequence (58). These data raise the question as to how Rep can target ch-19 among other RBE sequences. Using an Epstein-Barr virus (EBV)-based shuttle vector system carrying sequences from ch-19, Linden et al. demonstrated that the trs site was also critical for AAV site-specific integration (29, 30). When the trs site was not present, targeting was lost, even though the RBE was present. The present study suggested that both sequences were essential for site-specific integration (the RBE and the trs sequences). The probability of identifying a RBE with the correct proximity of a trs site would suggest a frequency of <6 × 10⁻¹¹/genome, thereby defining a unique sequence in the human genome (54). While these studies identify ch-19 cis elements required for AAV targeted integration and suggest why this reaction is specific, how Rep carries out this reaction remains unclear.Critical to any model of AAV Rep-mediated targeted integration is the ability to recognize the ch-19 target sequence among other potential RBE sequences. Though Rep can bind many degenerate sequences, the actual definition of what constitutes an RBE is somewhat unclear. Random oligonucleotide selection demonstrated that the RBE could be defined as an 8-bp sequence: 5′-GAGYGAGC-3′ (5). However, it was shown by methylation interference assays that the RBE was an 18-bp core sequence and that any mutation within this sequence would significantly affect Rep binding (42). Also, the report by Wonderling and Owens (58) demonstrated that the RBE oligonucleotides derived from the BLAST search contained mutations in this 18-bp core sequence but still bound better to the MBP-Rep68 than to the ch-19 RBE. Depending on the definition of an AAV RBE, the copy number present in the human genome (GAGYGAGC = 200,000 copies/genome, whereas 18-bp core = 1 copy/genome) could significantly impact the ability of Rep to identify its target locus.Based on the above information, the number of RBE sequences in the human genome, how Rep discriminates between these and the ch-19 target locus RBE sequence, and how Rep interacts with all sites and still facilitates targeted integration within a fixed time frame become of significant importance. In this study, we evaluated the role of alternative RBEs in the human genome and how these sequences might impact the ability of Rep to target the locus on ch-19. Using a filter-binding assay and a highly purified source of Rep68 protein, we established that genomic DNA will compete efficiently against a ch-19 target sequences. In this assay, a minimum Rep binding site of 8-bp in the context of large DNA fragments demonstrated competition, suggesting that as many as 200,000 potential binding sites may exist in the human genome. Filter-binding analysis of genomic DNA successfully retained ch-19 target sequences, as well as a cellular RBE identified by BLAST analysis, corroborating the competition results. Electron microscopy (EM) analysis was utilized to distinguish possible differences between Rep protein DNA interaction with ch-19 RBE compared to a minimum 8-bp RBE sequence. Identical multimeric Rep protein DNA complexes, which spanned about 60 bp, assembled on ch-19 target DNA, as well as a minimum RBE site, but never on heterologous DNA lacking these sequences. At a high Rep concentration, protein DNA looping structures were detected, but no evidence for paranemic structures were observed. In vivo analysis of Rep protein levels in a latent infection demonstrated approximately 1 to 4,000 copies/cell. Analysis of Rep expression in non-virus-infected cells demonstrated DNA rearrangement of the ch-19 target sequence, suggesting that this locus is a hot spot for Rep-induced DNA amplification and rearrangement that most likely influences AAV targeted integration. Finally, generation of an animal model carrying the human ch-19 sequence at the mouse hypoxanthine phosphoribosyltransferase (HPRT) locus facilitated AAV Rep-mediated targeted integration and corroborates the importance of the ch-19 RBE-trs sequence.     

A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.

The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.     

Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization.

We have used differential cell extraction and conventional chromatography to separate and partially purify the four adeno-associated virus (AAV) nonstructural proteins Rep78, Rep68, Rep52, and Rep40. In the cytoplasmic extracts Rep52 and Rep40 were present in greater abundance than Rep68 and Rep78, with Rep78 being the least abundant. In nuclear extracts the four Rep proteins were approximately equal in abundance. Regardless of the subcellular fraction examined, three of the Rep proteins (Rep78, Rep68, and Rep40) consisted of two protein species with slightly different mobilities during polyacrylamide gel electrophoresis. In contrast, Rep52 consisted of only one protein species. Both Rep78 and Rep68 were capable of binding efficiently to AAV terminal hairpin DNA substrates, but we could not detect site-specific DNA binding by Rep52 and Rep40. Like Rep68, Rep78 had both an ATP-dependent trs endonuclease and a DNA helicase activity. Both Rep78 and Rep68 cut the terminal AAV sequence at the same site (nucleotide 124). The binding, trs endonuclease, and DNA helicase activities comigrated during sucrose density gradient centrifugation with a mobility expected for a monomer of the protein, suggesting that the three biochemical activities were intrinsic properties of the larger Rep proteins. The chromatographic behavior and the DNA-binding properties of the four Rep proteins identified at least two domains within the rep coding region, an exposed hydrophobic domain within the C-terminal end (amino acids 578 to 621) and a region within the N terminus (amino acids 1 to 214) which was necessary for binding to the terminal repeat sequence. No site-specific nuclease activity was seen in the presence of nucleotide analogs ATP-gamma-S or AMP-PNP, suggesting that ATP hydrolysis was required for the endonuclease reaction. Furthermore, although ATP was the only cofactor which would support the trs endonuclease activity of Rep78, Rep68 nuclease activity was seen in the presence of several other nucleotide cofactors, including CTP, GTP, and UTP.     

Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.     

A biochemical characterization of the adeno-associated virus Rep40 helicase

The human adeno-associated virus (AAV) has generated much enthusiasm as a transfer vector for human gene therapy. Although clinical gene therapy trials have been initiated using AAV vectors, much remains to be learned regarding the basic mechanisms of virus replication, gene expression, and virion assembly. AAV encodes four nonstructural, or replication (Rep), proteins. The Rep78 and Rep68 proteins regulate viral DNA replication, chromosomal integration, and gene expression. The Rep52 and Rep40 proteins mediate virus assembly. To better understand Rep protein function, we have expressed the Rep40 protein in Escherichia coli and purified it to near homogeneity. Like the other Rep proteins, Rep40 possesses helicase and ATPase activity. ATP is the best substrate, and Mg2+ is the most efficient divalent metal ion for helicase activity. A Lys to His mutation in the purine nucleotide-binding site results in a protein that inhibits helicase activity in a dominant negative manner. Rep40 unwinds double-stranded DNA containing a 3' single-stranded end, or blunt end, unlike the Rep68 and Rep52 enzymes, which have a strict requirement for DNA duplexes containing a 3' single-stranded end. Values for KATP in the ATPase assay are 1.1 +/- 0.2 mM and 1.2 +/- 0.2 mM in the absence and presence, respectively, of single-stranded DNA. Values for Vmax are 220 +/- 10 and 1,500 +/- 90 nmol/min/mg in the absence and presence, respectively, of single-stranded DNA. These studies provide the first enzymatic characterization of the AAV Rep40 protein and elucidate important functional differences between the AAV helicases.