| Chemical carcinogens can cause modifications or damages on DNA directly. If these damages are not repaired properly, they result in mutagenesis and carcinogenesis.However, organism can react to the stress actively and the mutagenesis is not a passive event. SOS response is employed in DNA damaging agents challenged E. co/i, to cope with abnormal conditions. It has also been demonstrated that SOS response dependent accumulation of mutation during replication of DNA that has not been attacked by exogenous DNA damaging factors can be seen in challenged cells, termed as SOS untargeted mutation. In E coli, DNA polymerases are key enzymes involved in two distinguished pathways contributing to untargeted mutagenesis. Replication of DNA by pol V(UmuC), in the presence of UmuD1, RecA and single strand binding protein (SSB), is highly mutagenic and exhibits a predominant mutation pattern of base transversion. Another error prone polymerase involved in untargeted mutagenesis is pol IV, encoded by dinB gene. SOS induced pol IV increases untargeted mutagenesis not only in A phage, but also in chromosome of E. coli, characterized as -1 frameshift mutation.Much less is known about untargeted mutagenesis in eukaryotes. We and others have reported evidences of untargeted mutagenesis in mammalian cells. Our previous work identified delayed mutation occurred at target supF tRNA gene in plasmid transfected into cells pretreated with low concentration of alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Direct damageon supF tRNA gene can be neglected because half-life of MNNG is 1.1 hour and the interval between treatment and transfection was as long as 12-24 hours. Therefore the mutagenesis is not lesion directed. Furthermore, we found the mutagenesis relies on the alteration on gene expression profile induced by MNNG treatment; and also, the expression of DNA polymerase P (Pol P) was proved increased after MNNG treatment.Pol p is normally regarded as the base excision repair (BER) polymerase. Its role in BER is un-replaceable and thereby is important in mammalian genomic stability. However, it is recently cognized to be associated with genetic instability, due to its error prone features, i.e. lack of 3'?' proofreading activity and low replication fidelity in vitro, distributive manner in DNA synthesis, poor ability to discriminate nucleotides at the level of binding and also the possibility of its expanded roles in long patch BER, NER, DNA replication and recombination at its excess state. Therefore, we set out to explore the possible role of over-expressed Pol p induced by MNNG in untargeted mutagenesis. The signaling pathways involved in mediating Pol P over-expression were also studied.DNA polymerase p was induced by MNNG treatment. In this research, the expression of DNA polymerase p was examined in MNNG treated cells, where untargeted mutation had been proved arisen. After been treated with MNNG for 2.5 hours, cells were incubated for 12,18 or 24 hours before been harvested. The amounts of Pol P were determined with Western blotting. Results showed a slightly elevated expression of Pol P in MNNG treated cells after 12-hour-incubation (65.1 arbitrary unit in MNNG treated cells and 50.8 in control). The differences were enlarged 6 more hours later (125.5 and 66.8, respectively). Because of its low fidelity, over-expressed error prone Pol P might introduce mutations on both damaged and un-damaged DNA templates by perturbing other polymerases with high fidelity. Thus, we hypothesized that Pol P might be involved in MNNG induced untargeted mutagenesis.Establishment of Pol p down-regulated cell line: To verify the hypothesis, a human Pol p down-regulated cell line was established using an antisense RNA strategy. A 358 bp Pol ft cDNA fragment framed with introduced restrict sites ofXho I (upstream) and Nhe I (downstream) was amplified by the method of RT-PCR, and then cloned into a TA-vector. Inserted Pol ft cDNA fragment was recovered from the recombinant vector by Xho I and Nhe I digesting, an...
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