Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiaeretrotransposon Ty3 with conformationally distinct nucleic acid duplexes

The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading f rame was purified and evaluated on conformationally distinct nucleic acid d uplexes. Purified enzyme migrated as a monomer by size exclusion chromatogr aphy. 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 d uplex remained nuclease accessible. The C-terminal portion of Ty3 RT encode s a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conserv ation of catalytically important residues in the RNase H domain, Fe2+ fails to replace Mg2+ in the RNase H catalytic center for localized generation o f hydroxyl radicals, again suggesting this domain may be structurally disti nct 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 step s 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.