Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats

Abstract

Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68A produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, A57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68A of approximately 8 x 10-10 M was determined for the wt ITR and 457 ITR probes. Truncation of 457 ITR to generate 428 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than 457 ITR. Extension of A28 ITR with nonspecific sequence restored the ability of MBP-Rep684 to bind to A28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the A57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep684. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep684 or wt Rep68 to hairpin ITR or A57 ITR was indistinguishable. However, the binding activity of MBP-Rep684 to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68A to the nonhairpin 457 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep684 binding, its presence does affect the trs cleavage activity of the protein.

Negative regulation of the adeno-associated virus (AAV) P5 promoter involves both the P5 rep binding site and the consensus ATP-binding motif of the AAV Rep68 protein

“…Our deletion analyses indicated that the P 5 promoter region between the TATA box and the transcription initiation site is important for the negative regulation by Rep68. This region is similar to the RRSs identified previously in the AAV ITR and in the AAV integration site in human chromosome 19, which contain three perfect GAGC repeats and are bound by Rep proteins (12,13,22,23,42,56). Although the P 5 RRS contains only one perfect GAGC box, it is bound by Rep68, as shown by the gel shift analyses.…”

“…3B). The effect of the length of the fragment in stabilizing Rep binding has also been observed with the linear ITR DNA by using a purified maltose-binding protein-Rep68 fusion protein (13). Consequently, all of the oligonucleotide pairs analyzed in band shift analyses were cloned into pBluescript and tested as larger fragments.…”

Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles

“…Within the ITR region, two elements have been described which are central to the function of the ITR, a GAGC repeat motif and the terminal resolution site (TRS). The repeat motif has been shown to bind Rep when the ITR is in either a linear or a hairpin conformation (15,16,44). This binding serves to position Rep68/78 for cleavage at the TRS, which occurs in a site-and strand-specific manner.…”

Adeno-associated Virus as a Mammalian DNA Vector

“…There are three salient features of the AAV ITR that are required for vector production: (i) a 16-nucleotide tetrameric repeat (GAGC) sequence that is specifically bound by the Rep68/78 proteins termed the Rep-binding element (RBE), (ii) a sequence within the ITR internal hairpin that serves to orient Rep68/78 towards its resolution site termed RBE' (45)(46)(47)(48)(49), and (iii) the terminal resolution sequence (trs), which is the site of the strand-specific Rep68/78 endonuclease activity (44,(50)(51)(52). The N-termini of the larger Rep proteins contain a DNA-binding domain, as well as strand-specific endonuclease activity, necessary for initiating replication during virion production and site-specific integration (46,(53)(54)(55)(56). The C-terminal portion of Rep68/78 is required for multimerization and confers helicase activity which, in part, extrudes a putative nicking stem within the ITR sequence (54,57,58).…”