Overexpression of DNA polymerase beta in cell results in a mutator phenotype and a decreased sensitivity to anticancer drugs.

DNA polymerase beta (pol beta) is the most error prone of all known eukaryotic DNA polymerases tested in vitro. Here, we show that cells overexpressing pol beta 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 beta. 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 beta overexpression in cancer predisposition and tumor progression during chemotherapy.

Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin.

In this study the role of nuclear architecture in nucleotide excision repair (NER) was investigated by gentle dismantling of the cell and probing the capability of chromatin to carry out repair in vitro. The rationale behind this approach is that compartmentalization of NER at nuclear structures would make the enzymatic activities refractory to extraction by buffers that solubilize cellular membranes. In order to obtain intact chromatin primary human fibroblasts were encapsulated in agarose microbeads and lysed in isotonic buffers containing the non-ionic detergent Triton X-100. Under these conditions the majority of cellular proteins diffuse out of the beads, but the remaining chromatin is able to replicate and to transcribe DNA in the presence of triphosphates and Mg2+. UV irradiation of confluent repair-proficient human fibroblasts prior to lysis stimulated the incorporation of deoxynucleotide triphosphates in Triton X-100-isolated chromatin, even under stringent lysis conditions. In addition, experiments with UV-sensitive xeroderma pigmentosum (complementation groups A and C) and Cockayne's syndrome fibroblasts (complementation group A) revealed that this repair synthesis was due to global genome repair activity. Transcription-coupled repair was only detectable in cells permeabilized by streptolysin O (SLO). Repair synthesis in Triton X-100-isolated chromatin amounted to 15% of the total repair synthesis as measured in SLO-permeabilized cells. To allow the detection of these activities in vitro, presynthesis complexes have to be formed in intact cells, indicating that chromatin from Triton X-100-lysed cells is unable to initiate NER in vitro. Our data indicate that the components involved in the resynthesis step of NER are tightly associated with chromatin. A substantial fraction of total proliferating cell nuclear antigen (PCNA), which is required for the resynthesis step in NER, has been reported to become Triton X-100 non-extractable and tightly associated with nuclear structures after UV irradiation of cells. We propose that Triton X-100-resistant repair synthesis might be mediated by this chromatin-bound fraction of total PCNA.

Overexpression of DNA polymerase beta sensitizes mammalian cells to 2',3'-deoxycytidine and 3'-azido-3'-deoxythymidine.

Mammalian DNA polymerase beta is a DNA repair enzyme expressed constitutively at a low level. In vitro, purified DNA polymerase (Pol) beta incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphosphate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate into DNA, causing chain termination. We have tested the possibility of enhancing the cytotoxicity of these chain terminators against mammalian cells by increasing the level of Pol beta. Chinese hamster ovary AA8 and murine melanoma B16 cell lines were stably transfected with rat pol beta cDNA under the control of a viral enhancer/promoter. We found that overexpression of Pol beta sensitized the cells to ddC and AZT. To confirm the role of this polymerase in this process, we prepared cell extracts from the control and Pol beta overexpressing Chinese hamster ovary cell lines and tested in vitro their capacity to incorporate ddC-triphosphate and AZT-triphosphate into DNA. We found that inhibition of DNA replication by both chain terminators was more pronounced when extracts from pol beta-transfected cells were used, providing a direct evidence of the involvement of Pol beta in the sensitization process. In addition, we showed that cotransfection with bacterial or viral thymidine/thymidylate kinase genes enhanced the Pol beta-mediated cytotoxicity of AZT, suggesting that phosphorylation and polymerization activities might be combined to potentiate their respective effects. These observations may be useful for improving therapeutic efficiency of DNA chain terminators.

Influence of DNA supercoiling on cisplatin toxicity in Escherichia coli K-12.

DNA supercoiling is known to modulate the activity of numerous promoters in vitro and in vivo. Moreover, it has been reported to modulate the rate of formation of cisplatin/DNA crosslinks in vitro. In order to address the question of how the topology influences CDDP toxicity in E. coli, three mutants with altered gyrase activity which led to a decrease of about 25% in superhelical density were studied. Mutant strains gyrA224 and gyrB225 showed similar sensitivity to CDDP as the parental strain while the gyrB226 mutant was resistant. This resistance was abolished in uvrA (excision-repair) and recA (recombination and SOS processes) mutant derivatives. Thus supercoiling might play a role as an indirect modulator of CDDP toxicity in bacteria by interfering with repair processes.

Multiple mechanisms of resistance to cisplatin toxicity in an Escherichia coli K12 mutant.

The mechanisms underlying cellular resistance to the antitumor drug cis-diamminedichloro-platinum(II) (CDDP) were studied in Escherichia coli K12. A bacterial strain (MC4100/DDP) was selected from the MC4100 wild-type strain after growth for four cycles in CDDP. MC4100/DDP bacteria showed a high level of resistance and exhibited various modifications including (1) a decrease in drug uptake and platinum/DNA binding which only partly contributed to resistance, (2) an increase in glutathione content not involved in the resistant phenotype, (3) an increase in DNA repair capacity. Resistance was unmodified by introducing a uvrA mutation which neutralizes the excision-repair pathway. In contrast, it was abolished by deletion of the recA gene which abolishes recombination and SOS repair but also by a mutation in the recA gene leading to RecA co-protease minus (no SOS induction). RecA protein was unchanged in MC4100/DDP but the expression of RecA-dependent gene(s) was required for CDDP resistance. The regulation of genes belonging to the SOS regulon was analysed in MC4100/DDP by monitoring the expression of sfiA and recA::lacZ gene fusions after UV irradiation. These gene fusions were derepressed faster and the optimal expression was obtained for a lower number of UV lesions in MC4100/DDP, suggesting a role of RecA co-protease activity in the mechanism of resistance to CDDP in this E. coli strain.

UV induction of excision repair enzymes detected in protein extracts from Schizosaccharomyces pombe.

Induction of genes and proteins after DNA damaging treatment is well documented in various biological systems. In order to monitor repair activity in Schizosacchromyces pombe, we adapted the biochemical assay that allowed specific quantification of excision repair in mammalian cells (Wood et al. 1988, Cell, 53, 97-106) to yeast-free extracts. Repair synthesis determined on UV-damaged plasmid DNA with S. pombe total protein extract relied on base excision repair and not nucleotide excision repair. Under conditions that allowed optimal repair activity, an enhanced repair synthesis was found with extract from yeast previously irradiated with UV light (254 nm). A 4-fold induction factor was obtained with 70 J/m2 irradiation dose after 40 min incubation post-irradiation. This base excision repair activity on UV photoproducts was transiently induced since it returned to the level of untreated yeast after about 2 hours post-irradiation.

Resistance to cisplatin in an E. coli B/r NalR mutant.

The effects of various mutations in DNA-repair processes have been reported to either enhance or decrease bacterial sensitivity to cis-diamminedichloroplatinum(II) (cis-DDP). In the search for other mutations affecting bacterial sensitivity to this antitumor compound, we tested the E. coli B/r BS80 mutant, which is resistant to nalidixic acid (NalR). This mutation maps in the topoisomerase II gene (gyrA subunit) and leads to cross-resistance to cis-DDP. The mechanism underlying the resistance phenotype was only partly due to decreased DNA platination. BS80 was cross-resistant to mitomycin C and, to a lesser extent, to UV light, while it was normally sensitive to MNNG. The mechanisms involved in cis-DDP and mitomycin C resistance were independent of uvrA (excision repair) and recA (SOS repair and recombination) gene expression. In contrast, UV resistance was dependent upon recA gene expression. Both the reversion to NalS in BS80 and the transduction of NalR in the parental wild type (F26) did not modify cis-DDP toxicity; in addition, platinated plasmids equally survived in BS80 and F26 strains. Hence, it is possible that selection of the NalR phenotype induced other mutation(s) than gyrA responsible for cis-DDP, mitomycin C and UV resistance and/or that lesions with a different toxic potential were introduced by cis-DDP into the BS80 and F26 chromosomes.

In vitro evolution of cisplatin/DNA monoadducts into diadducts is dependent upon superhelical density.

DNA binding of antitumor platinum(II) compounds accounts for cellular toxicity. Binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to DNA involves the transient presence of monoadducts which evolve in a second phase into difunctional lesions which are far more toxic than the monoadducts. Temporal control of the monoadducts half-live is at least dependent upon the chemical nature of the cis-platinum derivative and the secondary structure of DNA. The effect of the degree of DNA superhelicity on the binding of cis-platinum derivatives as well as on the evolution of monofunctional adducts has been addressed on plasmid DNA. The rate of platination was not affected by the degree of DNA superhelicity. Similarly, when the evolution of the lesions was complete, no variation of toxicity was found with different populations of topoisomers, as determined by bacterial transformation efficiency. In contrast, when the kinetic of difunctional lesions formation was controlled in vitro, we observed a higher rate of formation on a supercoiled plasmid by comparison with a relaxed one. This result suggests that platinum-DNA adduct toxicity could be modulated by the topology of the chromosome.