| DNA is the carrier of genetic information, and the integrity and stability of genomic DNA is very meaningful for the live and the normal physical activity of cells. DNA polymerase β (Polβ) is a key enzyme in DNA base excision repair (BER) pathway, and is an important factor for maintaining genome integrity and stability. R137Q, one of DNA Polβ variants identified in tumors, as an arginine to glutamine substitution, was shown to lower polymerase activity and further to impair its DNA repair capacity. However, the exact functional deficiency of this polymorphism in vivo is still unknown.We would conduct the experiments from the aspect of embryo development, we had the female and male Polβ R137Q Knockin homozygous mice mated, after that we anatomized the E15.5 day mice embryos to get the tissues and mouse embryonic fibroblast cells of wide type, PolβWI/R137Q and R137Q embryos, we found an interesting thing that R137Q embryos were smaller than WT embryos, this let us consider that if any problems exists in the period of embryo development, due to cells growth process in embryo period is different from adult cells, we just speculated that cells in the embryo period have slower proliferation and higher apoptosis rate. And the results suggested that decreased levels of cell proliferation and increased levels of cell apoptosis were detected in the mutant mouse embryos. At the same time, found the expression of phosphorylated CHK1(Cell cycle checkpoint kinase 1) in R137Q MEFs was more than wide type tested by western blot, furthermore, the result demonstrated that R137Q MEFs have slower proliferation.Given that R137Q catalyzes DNA synthesis at slower rate than wide type, we speculated that unfilled gaps could lead to more chromosomal aberrations in R137Q MEFs. Therefore, wide type and R137Q MEFs were treated with colchicine, we analyzed the chromosomal morphology and found that there were more chromosomal breaks existed in R137Q MEFs. Many types of chromosomal aberrations arise from DNA breaks. We hypothesized that cells expressing only R137Q protein would be unable to completely fill all small gaps that arise in DNA during BER, leading to the accumulation of DNA breaks. To test the hypothesis BER assay was carried out and the results indicated that DNA repair efficiency was impaired with R137Q’s lower polymerase activity. DNA double-strand breaks were presented at significantly higher levels in the homozygous mutant R137Q versus WT and heterozygous PolβWT/RI37Q by Western Blot and immunofluorescence assay. However, the higher apoptosis ratio of R137Q MEFs might be caused by the deficient capacity of DNA repair so that lead to cells death. If DNA double-strand breaks could not be completely repaired. The results implied that DNA double helix structure may be damaged by some breakages that cannot be repaired successfully, the genomic DNA integrity and stability has been threaten.However, there is no obvious phenotype’s difference between R137Q and WT cells when cultured in the plates, we are curious that how to react when exposed to the environmental stimulation like DNA damaging reagents, DNA damaging reagents like alkylating agents and oxidizing agents can destroyed the structure of nucleotide, and DNA base alkylation and oxidization result in the base cannot bind with other elements that is important to form the complete structure of DNA. Eventually, it would ruin the structure of DNA double helix, and cells would die. Our results show that R137Q MEFs have more DNA double-strand breaks and increased apoptosis ratio. This means R137Q MEFs are more sensitive to DNA damaging reagents such as methyl methanesulfonate (MMS) and H2O2.Above all, our results indicate Polβ is essential for genomic stability and embryo development. This study make further understanding about how Polβ works in tumor cells or other diseases, and is meaningful for the prevention and therapy of DNA repair relative diseases. |
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