Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

1997 ◽  
Vol 71 (7) ◽  
pp. 4997-5002 ◽  
Author(s):  
G W Holzer ◽  
F G Falkner
Keyword(s):  
Dna Repair ◽  
Vaccinia Virus ◽  
Cell Line ◽  
Dna Glycosylase ◽  
Repair Enzyme ◽  
Uracil Dna Glycosylase

A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine

1992 ◽  
Vol 12 (12) ◽  
pp. 5536-5540
Author(s):  
R J Boorstein ◽  
L N Chiu ◽  
G W Teebor
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Mammalian Cell ◽  
Dna Glycosylase ◽  
Mammalian Cell Line ◽  
Repair Enzyme ◽  
Thymidine Analog ◽  
Large Numbers ◽  
Mechanism Of Toxicity ◽  
Base Excision

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.

Multiple microRNAs may regulate the DNA repair enzyme uracil-DNA glycosylase

DNA Repair ◽  
2013 ◽  
Vol 12 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Siv A. Hegre ◽  
Pål Sætrom ◽  
Per A. Aas ◽  
Henrik S. Pettersen ◽  
Marit Otterlei ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Glycosylase ◽  
Repair Enzyme ◽  
Uracil Dna Glycosylase

scholarly journals Molecular cloning of human uracil-DNA glycosylase, a highly conserved DNA repair enzyme.

1989 ◽  
Vol 8 (10) ◽  
pp. 3121-3125 ◽  
Author(s):  
L.C. Olsen ◽  
R. Aasland ◽  
C.U. Wittwer ◽  
H.E. Krokan ◽  
D.E. Helland
Keyword(s):  
Dna Repair ◽  
Molecular Cloning ◽  
Dna Glycosylase ◽  
Repair Enzyme ◽  
Uracil Dna Glycosylase

scholarly journals Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

2003 ◽  
Vol 77 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Frank S. De Silva ◽  
Bernard Moss
Keyword(s):  
Dna Replication ◽  
Vaccinia Virus ◽  
Cell Line ◽  
Essential Role ◽  
Cultured Cells ◽  
Catalytic Site ◽  
Dna Glycosylase ◽  
Uracil Dna Glycosylase ◽  
Vaccinia Viruses

ABSTRACT Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.

scholarly journals A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine.

1992 ◽  
Vol 12 (12) ◽  
pp. 5536-5540 ◽  
Author(s):  
R J Boorstein ◽  
L N Chiu ◽  
G W Teebor
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Mammalian Cell ◽  
Dna Glycosylase ◽  
Mammalian Cell Line ◽  
Repair Enzyme ◽  
Thymidine Analog ◽  
Large Numbers ◽  
Mechanism Of Toxicity ◽  
Base Excision

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.

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