INTERACTION OF THE ADENOASSOCIATED 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 ste m region of the adeno-associated virus terminal repeat (TR) and compar ed its affinity with that of the complete hairpinned TR for pure Rep68 . Both the A stem and the complete TR substrates produced a complex pa ttern of protein-DNA complexes in which at least six different bound s pecies 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 experiment s also suggested that the average number of Rep molecules per TR subst rate 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 differe nce was seen in the mobility or the pattern of the protein-DNA complex es 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 com plex rather than the actual number of Rep molecules per DNA molecule. At least some of the difference in stability of the two kinds of compl exes was due to the fact that the dissociation rate of the A stem subs trate from the protein-DNA complexes was approximately fourfold faster than that of the complete TR. The dissociation rate curves for both s ubstrates, 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 sub stitution 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 t he 25-bp sequence of the A stem that had been identified in the accomp anying 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 mutan ts completely abolished binding to the A stem substrate but only parti ally reduced binding in the context of the complete hairpinned TR. Fur thermore, neither mutant altered the pattern of Rep-DNA complexes prod uced. The analysis of these mutants supported the idea that at least t hree recognition elements which are involved in trs endonuclease activ ity are present in the TR: the A stem binding sequence, the secondary structure of the B and C palindromes, and the trs site. The results al so suggested that the 25-bp A stem binding sequence probably did not c onsist of multiple, independent Rep binding motifs but represented one binding motif. Finally, our results suggested that a number of differ ent Rep-DNA complexes can be stably formed and that some of these cont ain more than one DNA molecule.