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                                             BIOINFORMATICS COLLOQUIUM
					        College of Science
                                              George Mason University
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                      A Stable Secondary Structure Near the Nicking Site for Adeno-Associated 
                                 Virus Type 2 Rep Proteins on Human Chromosome 19
 
                                             Roland A. Owens, Ph.D.

                                       Chief, Molecular Biology Section
                                                 LMCB/NIDDK
                                        National Institutes of Health 
                                   U.S. Department of Health and Human Services

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' [slash indicates 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.