PMID: 9187238May 23, 1997Paper

Characterization and mutational studies of equine infectious anemia virus dUTPase

Biochimica Et Biophysica Acta
H ShaoS L Payne

Abstract

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ...Continue Reading

Citations

Oct 8, 2008·Accounts of Chemical Research·Béata G Vértessy, Judit Tóth
Aug 13, 2011·Proceedings of the National Academy of Sciences of the United States of America·Ildikó PécsiJudit Tóth
Jul 24, 2008·Avian Diseases·Li-chan ZhaoTao Sun
Jul 28, 2004·Journal of Molecular Biology·Sum ChanMichael R Sawaya
Sep 5, 2013·The Biochemical Journal·Glyn R HemsworthKeith S Wilson
Aug 12, 2015·Retrovirology·Amnon Hizi, Eytan Herzig
Jun 7, 2008·Retrovirology·Joséphine SireStéphane Priet
Oct 16, 2016·Journal of the American Chemical Society·Gergely N NagyEdina Rosta
Mar 21, 2001·FEBS Letters·J NordT Drakenberg
Jan 19, 2020·Journal of Molecular Biology·Emma ScalettiPål Stenmark

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