IDENTIFICATION OF LINEAR DNA-SEQUENCES THAT SPECIFICALLY BIND THE ADENOASSOCIATED VIRUS REP PROTEIN

We have used baculovirus-expressed Rep68 that has been purified to hom ogeneity to reexamine the binding properties of the Rep protein. We fi nd that Rep68 is capable of binding to a linear DNA sequence that is c ontained within a 25-bp sequence of the A stem of the adeno-associated virus (AAV) terminal repeat proximal to the B and C palindromes. This has been shown conclusively by demonstrating that Rep68 could specifi cally bind to a synthetic oligonucleotide containing the 25-bp region in the absence of the other sequences within the terminal repeat. Rep7 8 was also capable of binding the A stem recognition element, as demon strated by the fact that a DNA affinity column containing the 25-bp se quence can be used to purify Rep78. The ability to recognize the linea r DNA sequence within the A stem provides a mechanism by which the Rep protein can be oriented on the terminal repeat so that only the corre ct strand is cub at the terminal resolution site (trs site) during ter minal resolution. In addition, computer analysis suggests that sequenc es similar to the A stem element are present within the three AAV prom oter regions. Electrophoretic mobility shift experiments clearly demon strate that the p5 promoter contains a Rep binding sequence. DNase pro tection experiments indicate that the Rep binding sequence within the p5 promoter is located between the YY1 initiator sequence and the TATA binding site. This position immediately suggests a mechanism by which the Rep protein could act as a repressor or a transactivator of p5 tr anscription by interacting with either YY1 or TBP. In addition, gel sh ift experiments suggest that the p19 promoter also contains a Rep bind ing site. The presence of Rep binding sites upstream of both promoters suggests that these sites may be involved in coordinate regulation of AAV transcription. In addition, we have identified a heterologous Rep binding sequence within pBR322 DNA. A comparison of the sequences wit hin the A stem, p5, and pBR322 binding sites suggests that a repeating GAGC motif is at least part of the Rep recognition sequence. In the a ccompanying report (D. M. McCarty, J. H. Ryan, S. Zolutukhin, X. Zhou, and N. Muzyczka, J. Virol. 68:4998-5006, 1994), we examine the relati ve affinity of Rep to the A stem site and the complete terminal repeat . Finally, we also have reexamined the ability of Rep68 and Rep78 to c ut at the trs site in substrates that do not contain the B and C palin dromes or any apparent secondary structure. Although higher amounts of enzyme are necessary, trs endonuclease activity can be detected on su ch substrates with both Rep68 and Rep78. This finding suggests that th e A stem recognition element and a functional trs site are sufficient for site specific cutting at the trs site and provides a mechanism by which dimer molecules, which are not expected to have hairpins, might be processed to monomer length during AAV DNA replication. It also mea ns that the AAV terminal repeat is a tripartite origin for DNA replica tion. For efficient function, three elements of the terminal repeat ar e necessary: the trs site, the A stem-binding element, and the B and C palindromes.