| Chemical carcinogen can lead to DNA damaging effects or non-DNA damaging effects (epigenetic effects). The former may induce DNA lesions and result in lesion-targeted mutation by translesion DNA synthesis pathway (TLS, also can be called DNA damage tolerance) if the lesion couldn't be effectively repaired. Mutation that occurs on undamaged DNA template is designated as untargeted mutation, which may be induced by both DNA damaging effects or non-DNA damaging effects.When bacteria E. coll. are exposed to environmental agents, such as UV light, SOS response will occur to avoid negative effect. As a result, untargeted mutation occurs due to the involvement of DNA polymerase IV and V. Pol IV alone mainly induces -1 frameshift mutagenesis, however, pol V plays a role in untargeted mutation in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol Ill's processivity beta, gamma-complex. The mutation spectrum induced of pol V is dominated by transversions (53%). The analysis of independentmutations reveals that the ratio between single-nucleotide substitutions and one-base deletions is about 1:2. However, the mechanism of eukaryotic untargeted mutagenesis is still unclear.Previous works in our laboratory revealed that epigenetic alteration occurs at the early stage after remove of low concentration MNNG treatment, including enhanced tryosine phosphorylation level of 45KD protein and 62KD protein, activation of JNK/SAPK and cAMP-PKA-CREB pathways and the activation of NF-KB. Meanwhile, differential gene expression can be detected in MNNG-treated mammalian cells, which is in a state of inducing untargeted mutagenesis. Further studies showed that DNA replication fidelity is decreased and there is an alteration of DNA polymerase spectrum when mammalian cells are attacked by MNNG. In addition, it is clear that the signal to activate certain signal transduction pathways is not from nucleus. Consequently, a hypothesis on mammalian untargeted mutation was given, that is, DNA polymerases with low DNA replication fidelity, as effectors, might be involved in mammalian untargeted mutation. When certain signal transduction pathways are activated by MNNG, differential gene expression is induced, and leads to activation of specific DNA polymerases, which decrease the DNA replication fidelity and finally result in mammalian untargeted mutagenesis. But it is not clear that which DNA polymerase plays a role in the process. In this study, the role of human REVS gene, which encodes the catalytic subunit of human DNA polymerase, in mammalian untargeted mutagenesis wasinvestigated, and the basic mechanism of transcriptional regulation of REVS was studied.Results showed that low concentration MNNG treatment led to upregulation of REVS gene expression at transcriptional level with in FL cells. After remove of MNNG, the REVS transcriptional level was significantly increased by 1.77-fold at 12h time point and 1.65-fold at 24h time point respectively as compared to DMSO control (p < 0.05). The alteration phase of REVS transcriptional level is consistent with occurrence phase of mammalian untargeted mutation induced by MNNG, which was observed previously in our laboratory. Further analysis of untargeted mutation in this study revealed that blockage of REVS gene function by antisense REVS mRNA had an anti-mutation effect and returned to the level of spontaneous mutation frequency, with significantly decrease of untargeted mutation frequency from 27.4+10-4 to 4.0+10-4 (p < 0.01). The blockage effect of antisense REVS mRNA in transgenic cell line FL-REV3" cells was confirmed by western blotting. Thus, in the first time, this study provides the evidence that DNA polymerase,is involved in MNNG-induced mammalian untargeted mutation.As the above-mentioned evidences, low concentration MNNG can induce early epigenetic alteration in mammalian cell, activate CREB, AP-1 and NF-KB. In this study, bioinformatic analysis found that the transcriptional binding sites for these transcriptional fa...
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