The Molecular Mechanism Of The Transcriptional Activation Of Human Ribonucleotide Reductase Small Subunit RRM2/p53R2Genes Induced By MNNG

N-methyl-N-nitro-N-nitrosoguanidine(MNNG), a mono-functional alkylating agent, has been known as a widespread environmental carcinogen, which could cause DNA lesion, mutation and even carcinoma. DNA damages induced by MNNG exposure can activate many DNA repair-related genes. Ribonucleotide reductase (RNR) plays a vital role in DNA synthesis and repair by catalyzing de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. The human ribonucleotide reductase is composed of two identical big subunits (RRMl) and two identical small subunits (RRM2or p53R2), which form two types of RNR, i.e. RRM1-RRM2and RRM1-p53R2. However, it is still unclear wether or how MNNG affect RNR to participate in cellular responses to the DNA damage.In this study, we have investigated the transcriptional changes and regulatory mechanisms of human RNR in cells after exposure to MNNG (1) We demonstrated that the expression of RRM2and p53R2, but not RRM1, was induced by MNNG through transcriptional regulation. At the same time, MNNG also induced the nuclear translocation of RRM2and p53R2.(2) By measuring the level of y-H2AX, cell cycle, and apoptosis after MNNG treatment, we showed that RRM2mainly contributed to the DNA repair while p53R2played a relative weaker role in it.(3) With DNA pull-down coupled with LC-MS/MS, ChIP-qPCR, and Western blot, we have found that the binding of E2F3, E2F1and NFY to RRM2promoter was markedly enhanced after MNNG exposure. The binding of E2F3and E2F1to RRM2promoter was enhanced by NFY which binding-site was adjacent to that of E2F. Immunoprecipitation/immnuoblotting analyses indicated that NFY could interact with E2F3in cells. Reporter assays showed that E2F3activated the promoter of RRM2more significantly than E2F1. In KB cell lines, over-expression of E2F3could increase the expression of RRM2thus reduced the level of y-H2AX while silencing of E2F3suppressed the up-regulation of RRM2induced by MNNG and increased y-H2AX.(4) Interestingly, E2F3level was increased by MNNG exposure mainly through posttranslational modification. At the same time, we found that the mRNA of NFYA and NFYB increased rapidly after MNNG addition. The increased NFY could translocate from cytoplasm to nucleus where it might regulate the transcription of RRM2together with E2F3.(5) We also observed that MNNG transactivated E2F3expression through ATR-CHK1-E2F3pathway, which in turn activated RRM2transcription and nuclear translocation to provide enough dNTPs for DNA repair. In addition, the upregulation of p53R2was dependent on not only p53but also E2F1activity., in comparison, the upregulation of RRM2was mainly relied on E2F3and NFY in response to MNNG exposure.In conclusion, these results suggest that RNR paticipates in the DNA damage repair induced by MNNG, which may play a pivotal role in maintaining genome stability. Importantly, we demonstrate that E2F3, NFY and their related pathways are responsible for the up-regulation of RRM2induced by MNNG It would be reasonable to conjecture that the abnormal regulation of RNR could lead to the mutation and carcinogenesis caused by environmental chemical carcinogens...