title: Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities.
abstract: In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Rausch
authors.forename: E J
authors.initials: EJ
authors.lastname: Arts
authors.forename: B M
authors.initials: BM
authors.lastname: Wöhrl
authors.forename: S F
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis
chemical_list: D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H