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dc.contributor.authorROTH, MONICA JUDITH
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 45-05, Section: B, page: 1355.
dc.description.abstractEnzyme reactions frequently proceed via covalent protein-substrate intermediates. The studies presented focus on two enzymes in which covalent catalysis is involved, the (phi)X174 gene A protein and vaccinia RNA: guanylyltransferase.;The (phi)X gene A protein is a bacteriophage encoded enzyme required for initiation and termination of single-stranded circular (SS(c)) viral DNA synthesis. The enzyme initiates DNA synthesis by nicking the supercoiled (phi)X replicative form (RFI) DNA at the viral (+) strand replication origin, and remains covalently bound to the 5'-terminus. After one round of synthesis, the displaced DNA strand is segregated as a unit length (+) SS(c) progeny molecule by the (phi)X A protein-mediated termination of SS(c) DNA synthesis. Subsequent SS(c) DNA synthesis requires the (phi)X A protein catalyzed reinitiation of DNA synthesis. Based on a model of covalent catalysis, the assumption was made that DNA molecules which had reinitiated a second round of (+) SS(c) synthesis would contain the (phi)X A protein covalently bound to the nascent DNA. In the experiments described here, incorporation of {lcub}(alpha)('32)P{rcub} deoxynucleoside triphosphates into the in vitro synthesized DNA, has permitted the isolation of a (phi)X A protein-{lcub}('32)P{rcub}-DNA complex. The results reported herein characterize the (phi)X A protein-{lcub}('32)P{rcub}-DNA complex in terms of (1) the proteins required for its formation, (2) the minimal DNA sequence at the (phi)X (+) strand replication origin sufficient for its formation, (3) structural analysis of the covalent protein-DNA linkage, and (4) function of the complex in the phage replication cycle, specifically in relation to the other known (phi)X A protein activities.;Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5' phosphate of diphosphate terminated RNA, generating the cap structure G(5')ppp(5')N-. The nucleotide transfer occurs in two partial reactions, during which the enzyme is covalently bound to a guanylate moiety. Herein, the nature of the enzymeguanylate complex is characterized. The covalent complex contains GMP bound to a lysine residue of the Mr = 95,000 subunit of vaccinia capping enzyme. Specifically, the linkage is a direct P to N bond between the (epsilon)-amino group of lysine and the phosphoryl group of GMP.
dc.publisherProQuest Dissertations & Theses

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