Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.
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PMID:DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells. 841 69

Vaccinia virus infection results in the synthesis of a protein that promotes joint molecule formation and strand-transfer reactions in vitro. We show here that this activity is also expressed by vaccinia DNA polymerase (gpE9L). Recombinant vaccinia polymerase was produced using a hybrid vaccinia/T7 expression system and purified to homogeneity. This protein catalyzed joint molecule formation and strand transfer in vitro in reactions containing single-stranded circular and linear duplex DNAs. The reaction required homologous substrates and magnesium ions and was stimulated by DNA aggregating agents such as spermidine HCl and Escherichia coli single-strand DNA binding protein. There was no requirement for a nucleoside triphosphate cofactor. The reaction ceased when approximately 20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantities of DNA polymerase (0.5-1 molecules of polymerase per double-stranded DNA end). Electron microscopy showed that the joint molecules formed during these reactions contained displaced strands and thus represented the products of a strand-exchange reaction. We also reexamined the link between replication and recombination using a luciferase-based transfection assay and cells infected with DNA polymerase Cts42 mutant viruses. These data substantiate the claim that there exists an inextricable link between replication and recombination in poxvirus-infected cells. Together, these biochemical and genetic data suggest a way of linking poxviral DNA replication with genetic recombination.
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PMID:Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions. 1032 61