DNA polymerase  (pol ) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol  cDNA have acquired a spontaneous mutator phenotype. By measuring the appearance of mutational events using three independent assays, we found that genetic instability increased in the cell lines that overexpressed pol . In addition, these cells displayed a decreased sensitivity to cancer chemotherapeutic, bifunctional, DNA-damaging agents such as cisplatin, melphalan, and mechlorethamine, resulting in enhanced mutagenesis compared with control cells. By using cell-free extracts and modified DNA substrates, we present data in support of error-prone translesion replication as one of the key determinants of tolerance phenotype. These results have implications for the potential role of pol  overexpression in cancer predisposition and tumor progression during chemotherapy.
The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by longpatch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase III␣. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.oxidative stress ͉ XRCC1 ͉ DNA ligases ͉ DNA single-strand breaks ͉ 8-oxoguanine
Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR ± ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR ± ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A signi®cant 3 ± 5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR ± ABL transfection induced an overexpression of DNA polymerase b, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase b in this process.
DNA polymerase β (Pol β) is the most inaccurate of the six DNA polymerases found in mammalian cells. In a normal situation, it is expressed at a constant low level and its role is believed to be restricted to repair synthesis in the base excision repair pathway participating to the genome stability. However, excess of Pol β, found in some human tumors, could confer an increase in spontaneous mutagenesis and result in a highly mutagenic tolerance phenotype toward bifunctional DNA cross‐linking anticancer drugs. Here, we present a hypothesis on the mechanisms used by Pol β to be a genetic instability enhancer through its overexpression. We hypothesize that an excess of Pol β perturbs the well‐defined specific functions of DNA polymerases developed by the cell and propose Pol β‐mediated gap fillings during DNA transactions like repair, replication, or recombination pathways as key processes to introduce illegitimate deoxyribonucleotides or mutagenic base analogs like those produced by intracellular oxidative processes. These mechanisms may predominate during cellular nonproliferative phases in the absence of DNA replication.—Canitrot, Y., Fréchet, M., Servant, L., Cazaux, C., Hoffmann, J.‐S. Overexpression of DNApolymerase β: a genomic instability enhancer process. FASEB J. 13, 1107–1111 (1999)
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