Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature- sensitive ubiquitin-activating enzyme (E1)

Hironobu Ikehata, Sumiko Kaneda, Fumiaki Yamao, Takeshi Seno, Tetsuya Ono, Fumio Hanaoka

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15 Citations (Scopus)

Abstract

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39°C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase α, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39°C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39°C, and more than 36 h at 39°C did not produce any further sensitization. Moreover, while the 39°C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Original languageEnglish
Pages (from-to)1484-1489
Number of pages6
JournalMolecular and cellular biology
Volume17
Issue number3
DOIs
Publication statusPublished - 1997 Mar

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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