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Journal of Virology, March 2005, p. 3544-3556, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3544-3556.2005

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

Ming Y. Jang,¹ OrLando H. Yarborough III,^1, Gary B. Conyers,¹ Peter McPhie,² and Roland A. Owens^1*

Laboratory of Molecular and Cellular Biology,¹ Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland²

Received 5 August 2004/ Accepted 22 October 2004

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.

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* Corresponding author. Mailing address: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8, Rm. 310, National Institutes of Health, Department of Health and Human Services, 8 Center Dr. MSC 0840, Bethesda, MD 20892-0840. Phone: (301) 496-3359. Fax: (301) 402-0053. E-mail address: ro6n{at}nih.gov.

Present address: Genetics Graduate Program, Yale University School of Medicine, New Haven, CT 06520-8005.

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Journal of Virology, March 2005, p. 3544-3556, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3544-3556.2005

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