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

Abstract

The Chinese hamster V79 cell mutant aphr4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase a (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase a 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 a-type polymerase: sensitivity to 0.2 M KCI, resistance to heat denaturation (450C for 15 min), an apparent Km of 5 FM for dATP, and an ability to copy poly(dT)(rA)10 but not poly(rA)J(dT)12.The crude extracts and purified DNA polymerase a from the mutant cells are not inhibited by aphidicolin (>0.6 pAM). The apparent Km for dCTP with DNA polymerase a is 1.0 ± 0.4 pM (mean ± SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent K. for dCTP of 10 ± 4 FM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.& 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 a may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.Three generic DNA polymerases in mammalian cells have been purified, characterized, and designated as a, A, and y (1). The roles of these enzymes in cellular metabolism have been difficult to identify unambiguously in the absence of mutant polymerases. Conversely, analysis of the phenotype of cells containing mutant polymerases might help to establish the contribution of DNA polymerases to the fidelity of DNA replication and repair and to determine the involvement of each polymerase in spontaneous and induced mutagenesis.Evidence accumulated over the past two decades suggests that DNA polymerase a (pol a) is primarily associated with DNA replication. (2). DNA pol a is the major activity present in replicating cells, increases during the DNA synthetic period of the cell cycle (3), and is induced when quiescent cells are stimulated to undergo DNA replication (4). Furthermore, at the time of DNA synthesis, there is evidence for the translocation of pol a from the cytoplasm to the nucleus (5, 6).Aphidicolin, a tetracyclic diterpenoid mycotoxin, specifically inhibits DNA pol a but not polymerase ( or y. The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8-11). There is controversy, however, concerning whether or not aphidicolin also inhibits DNA repair synthesis (12-14).We have previously reported (15,16) the s...

An improved method for isolation of mutator mutants from mouse FM3A cells and their characterization.

“…None of the cell clones had any alteration in sensitivity to these agents. The results suggested that these mutator mutants were different from the mutator mutants reported by Liu et al (9), which showed hypersensitivity to UV.…”

“…These cell clones were first isolated as mutants with altered sensitivity to inhibitors of DNA replication, such as arabinofuranosylcytosine (AraC) or aphidicolin. In addition to these mutator mutants, Liu et al (9) recently reported that one of the mutants resistant to aphidicolin had altered DNA polymerase a. They showed that the mutant DNA polymerase was resistant to aphidicolin and had a decreased Km for dCTP.…”

A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication

“…At this point, the reaction products were chased with a 300-fold excess of cold dCTP followed immediately by various amounts of aphidicolin. (Aphidicolin is an inhibitor of human pol alpha, delta, and epsilon, whose presence leads to chain termination [13,16]. In an Ad-infected extract, the presence of 0.25, 0.5, and 1.0 M aphidicolin inhibits total AAV DNA replication by 68, 83, and 93%, respectively [data not shown].)…”

Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA methyltransferase in human cancer

“…Abnormalities in DNA repair and replication have also long been considered as key elements in the genesis of mutations. Several mechanisms can be invoked to contribute to the infidelity of DNA synthesis, including imbalances in deoxynucleotide triphosphate pools, mutations in DNA polymerase-a and slippage of DNA polymerase at nucleotide repeats (Liu et al, 1983;Phear et al, 1987;Kunkel et al, 1993). However, the importance of each mechanism and gene(s) involved is often unknown.…”