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 ␣, 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 ubiquitinconjugating 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 ubiquitinconjugation system contributes to DNA repair in mammalian cells.Mutations that lead to temperature-sensitive (ts) phenotypes usually occur in genes essential for cell viability. Since DNA repair plays a critical and basic function to preserve the genetic information of cells against external and internal DNA-modifying agents, it is logical to assume that some ts mutations fall within DNA repair genes. We isolated ts mutants from the mouse mammary carcinoma cell line FM3A and classified them into 11 genetic complementation groups (8) comprising three groups of defective genes, each of which encoded DNA polymerase ␣ (Pol ␣), ubiquitin-activating enzyme (E1), or p34 cdc2 kinase. There are indications that these three gene products contribute to cellular DNA repair mechanisms. First, a study with DNA polymerase inhibitors suggested that Pol ␣ is a repair synthesis enzyme, in addition to its role in DNA replication (44). Second, a rad6 DNA repair mutant of Saccharomyces cerevisiae is defective in UBC2 (14), a ubiquitin-conjugating enzyme (E2), to which E1 transfers ubiquitin. This suggests relevance to DNA repair of the ubiquitin-conjugation system in which E1 and E2s cooperate to mark proteins with ubiquitins as substrates for intracellular nonlysosomal protease complexes (10). Third, a number of cellular responses to DNA-damaging agents in eukaryotes are mediated through cell cycle arrest or delay (7, 25), which is regulated by the cyclin-cdk systems in which p34 cdc2 kinase is a key enzyme (30).In the present study, we attempted to evalu...