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PMID: 6402775 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 80 ·No. 3 ·1983-02-00 ·Pages 797-801

Liu PK, Chang CC, Trosko JE, Dube DK, Martin GM, Loeb LA

Abstract

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.

MeSH Terms
Animals Aphidicolin Cells, Cultured Cricetinae DNA Replication DNA-Directed DNA Polymerase/genetics Deoxyribonucleotides/metabolism Diterpenes/pharmacology Drug Resistance Kinetics Mutation Nucleic Acid Synthesis Inhibitors
Chemicals
Deoxyribonucleotides Diterpenes Nucleic Acid Synthesis Inhibitors Aphidicolin DNA-Directed DNA Polymerase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Liu P K
Chang C C
Trosko J E
Dube D K
Martin G M
Loeb L A
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41 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.80.3.797
Published
1983-02-00
Pages
797-801
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC393467
Subset
IM
Grants
NIA NIH HHS · AG 00057 · United States
NCI NIH HHS · CA 11525 · United States
NCI NIH HHS · CA 12818 · United States
External Links
PubMed source DOI: 10.1073/pnas.80.3.797 PMC Full Text View journal details
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