A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins.
The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.
Wu
J
J
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 310, 8 Center Drive, MSC 0840, Bethesda, Maryland 20892-0840, USA.
Davis
M D
MD
Owens
R A
RA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006801:Humans; D066298:In Vitro Techniques; D009154:Mutation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020079:Terminal Repeat Sequences; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004247:DNA; D004720:Endonucleases