title: Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.
abstract: Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.
authors.affiliation: Molecular Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
authors.forename: J A
authors.initials: JA
authors.lastname: Chiorini
authors.forename: M D
authors.initials: MD
authors.lastname: Weitzman
authors.forename: R A
authors.initials: RA
authors.lastname: Owens
authors.forename: E
authors.initials: E
authors.lastname: Urcelay
authors.forename: B
authors.initials: B
authors.lastname: Safer
authors.forename: R M
authors.initials: RM
authors.lastname: Kotin
mesh_terms: D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002888:Chromosomes, Human, Pair 19; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D000229:Dependovirus / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; C068734:rep proteins, Adeno-associated virus 2; D003851:Deoxyribonucleases; D004265:DNA Helicases