title: Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes.
abstract: The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe(2+) fails to replace Mg(2+) in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.
authors.affiliation: Human Immunodeficiency Virus Drug Resistance Program, Division of Basic Sciences, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Rausch
authors.forename: M K
authors.initials: MK
authors.lastname: Grice
authors.forename: M
authors.initials: M
authors.lastname: Henrietta
authors.lastname: Nymark-McMahon
authors.forename: J T
authors.initials: JT
authors.lastname: Miller
authors.forename: S F
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase