, Volume 7, Issue 2, pp 235-253

Isolation and characterization of a UV-sensitive hypermutable aphidicolin-resistant Chinese hamster cell line

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Abstract

Aphidicolin is a specific inhibitor of DNA polymerase α and blocks DNA synthesis in vivo. The inhibition of purified α-polymerase has been shown to be competitive with dCTP but not with the other three deoxynucleoside triphosphates (dNTPs). In order to study the various roles that the α-polymerase might play in DNA replication and/or repair, we have attempted to isolate Chinese hamster V79 cells that are resistant to aphidicolin. Four resistant mutants were isolated from BrdU-black light- and UV-mutagenized cells. None of the mutants isolated contains an α-polymerase that is resistant, in crude extract measurements, to aphidicolin. Three mutants isolated, however, were found to be resistant to araC. Two mutants tested were found to be sensitive to cytidine and have elevated levels of dCTP or all 4 dNTPs. These results indicate that they are nucleotide pool mutants instead of α-polymerase mutants. One mutant, aphr-4, is characterized by the following: (1) high level of dCTP; (2) thymidine (or CdR, UdR, auxotrophic; (3) sensitive to thymidine (and AdR, GdR); (4) slow-growing; (5) cytidine sensitive; (6) UV sensitive and hypermutable at the ouabain-resistant locus; and (7) a ninefold increase in frequency of chromatid gaps and breaks when cells are exposed to BrdU- containing medium. Revertants of aphr-4 which are partially aphidicolin-resistant and retain the first three characteristics listed above, but not the others, have been isolated. The appearance of this type of revertant indicates that either aphr- 4 or its “revertant” is a double mutant.