Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19

Abstract

Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5-GGCGGCGGT/TGGGGCTCG-3 [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.Adeno-associated virus serotype 2 (AAV-2) is a human parvovirus. AAV-2 normally requires a helper virus, such as an adenovirus or herpesvirus for productive infection (7). In the absence of helper virus, AAV-2 can establish a latent infection by integrating its genome into the host DNA. AAV-2 preferentially integrates its DNA into a 4-kb region of human chromosome 19 (19q13-qter), designated AAVS1 (27-30). Subsequent infection by helper virus leads to rescue and productive infection. Preferential integration into AAVS1 requires the Rep68 or Rep78 protein (Rep68/78), encoded by AAV-2, as well as specific DNA sequences within the virus genome and AAVS1 (3,11,35,42,47,55,57,59). Binding sites for Rep68/78 within both the viral DNA and AAVS1 appear to be required (35,42,55,59). These sites, called Rep recognition sequences (RRSs), are comprised of imperfect repeats of the sequence 5Ј-GCTC-3Ј or its complement, 5Ј-GAGC-3Ј (11,20,59). Rep68/78 can form a bridge between RRS-containing DNAs that may facilitate preferential integration (34, 59).Rep68 and Rep78 also have a nucleoside triphosphate-dependent, strand-specific, site-specific endonuclease (nicking) activity (22,23). A Rep68/78 nicking site is called a terminal resolution site (trs) because of the role of such nicking sites in AAV-2 replication (22, 50, 52). Nicking requires both a specific sequence flanking the trs and a nearby...

The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element

“…Previous analyses on the viral ITR demonstrated that a potential stem-loop was present at the trs and that the extrusion of this structure was mediated by the ATPdependent helicase activity of Rep (3,4). A secondary structure was also recently described near the trs within the AAVS1 locus (20). In the present study, we demonstrated that deletion of the nucleotides forming the stem of the hairpin impaired p5 replication.…”

“…Two of these elements, the Rep-binding site and the trs, are also present within the chromosome 19 AAVS1 locus, where wild-type AAV-2 was shown to site-specifically integrate (21,26,43). More recently a secondary structure was also described near the trs of AAVS1 (20). Besides its role during AAV-2 site-specific integration, this chromosomal element was also shown to be able to replicate in the presence of Rep (55).…”

A novel G‐quadruplex motif modulates promoter activity of human thymidine kinase 1

“…Interestingly, the nick site for adenoassociated virus type 2 on human chromosome 19 was observed to fold into a quadruplex structure. The sequence, GGCGGCGGTTGGGGCTCG, indicates a quadruplex motif comprising two G‐tetrads in the core [41]. In addition to this, RNA sequences containing runs of two guanines could also form quadruplex motifs and be physiological targets of the fragile X mental retardation protein [40].…”

Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

“…Another ITR recognition sequence, RBEЈ, increases the binding affinity (68,89) and stimulates the Rep helicase activity that is necessary for initiating site-specific nicking (16,48). Helicase activity is believed to be necessary to unwind the duplex trs and extrude a stem-loop structure, which is the subsequent substrate for the nicking reaction (17,50). Strand-specific nicking of the ITR occurs at the trs, 3Ј-CCGGT/TG-5Ј, which results in a phosphotyrosyl linkage between Rep and the nicking site (17,48,99).…”