BINDING-SITES FOR ADENOASSOCIATED VIRUS REP PROTEINS WITHIN THE HUMANGENOME

The Rep proteins of adeno-associated virus type 2 (AAV) are known to b ind to Rep recognition sequences (RRSs) in the AAV inverted terminal r epeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV geno me into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration Locus. In an attempt to identify potential alternate inte gration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAV S1 separately as query sequences and identified 18 new RRSs in or flan king the genes coding for the following: tyrosine kinase activator pro tein I (TKA-1); colony stimulating factor-1; insulin-like growth facto r binding protein 2 (IGFBP-2); histone H2B.1; basement membrane hepara n sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is invoiced in the chromosomal translocation t (9:11)(p22:q23); the beta(B) subunit of the hormone known as inhibin; interleukin-2 en hancer binding factor; an endoplasmic reticulum-Golgi intermediate com partment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible fac tor, also known as midkine; a breast tumor autoantigen; a growth-arres t- and DNA-damage-inducible protein called gadd45; the cyclin-dependen t kinase inhibitor called KIP2, which inhibits several G(1) cyclin-cyc lin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRAC1). RRSs were also identified in a newly discovered o pen reading frame on chromosome 10 and in the ERCC1 locus on human chr omosome 19. The ability of a maltose binding protein-Rep68 fusion prot ein to bind to these sequences was confirmed by electrophoretic mobili ty shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.