The Cellular Localization Of Human Wild Type DNA Polymerase β And The Effect Of Biological Function On EC9706 Cell

Background and objective:Tumor is a consequence that the mechanism of cell differentiation proliferation and death is out of the way .It is the premise of cell malignant transformation that the abnormity of DNA damage, gene structure and the change of expression and function of oncogene and/or cancer suppressive gene ,which is result from the abnormity of DNA damage and gene structure. However ,not all damages lead to gene mutation because DNA repair system of cell can clean DNA damages out and repair them. Base-excision repair(BER) is one of vital ways to clean DNA damages out and that DNA polymerase β(DNA pol β) is a key enzyme in BER process. DNA pol β is involved in repairing DNA damages resulted by ROS oxidation or deamination of cytosine and met-cytosine. It also may be involved in repairing alkyl bases damage resulted by exterior factors such as nitrosamine .Therefore , DNA pol β can decrease gene mutations resulted from DNA damages .On the basis of the peculiar functions of DNA pol β ,we constructed a recombined enhanced green fluorescent protein expression vector carrying wild type DNA pol β gene in esophageal tissue ,and then transfected recombinants into EC9706 cell by lipofectamine method .The fusion protein was expressed in EC9706 cell .We observed the change of biological function of transfected cell to study indirectly the repair function of DNA pol β.Methods:The fragment of wild type DNA pol β gene was amplified by PCR method with plasmid pcDNA3.1-polβ as a template and cloned into pGEM-T vector by T-A clone method. And then, it was inserted directionally into a eukaryotic expression vector pEGFP-C3.The recombinant pEGFP-C3-polβ was obtained .Restricted enzyme analyzing, PCR technique and sequencing were employed to identify the recombined plasmid. The recombinant was transfected into EC9706 cells by lipofectamine method .The stable expressing EC9706-wtpolβ was established through G418 screening. The localization of DNA pol β gene-encoded protein was observed by fluorescent converted microscope. The expression of mRNA level was detected by RT-PCR method. The growth curve was drawn by MTT method and cell cycle was examined by flow cytometer .The effect of biological function of EC9706 cell transfected DNA pol β was analyzed. All data were analyzed statistically.Result:1. It was proved that the sequence of the wide type recombinant was correct and it was transfected into EC9706 cell successfully .2. The localization of wtDNA Pol p protein displayed that pEGFP-C3 and pEGFP-C3-polβ proteins expressed GFP and they were distributed in the whole cell and mostly inside the nuclear at 24 hour, respectively .There was significant difference between these two groups .And the expressing account of wtDNA Pol P protein was significantly higher than untransfected group.3. The proliferous speed of EC9706-wtPolβ was significantly slower than EC9706-neor and EC9706. (P<0.05)。 Furthermore, the cell number of S period was decrease in EC9706-wtPolβ (P>0.05) .Conclusion:1. The recombined expression vector (pEGFP-C3-polβ) is constructed successfully.2. The localization of exogenous wtDNA Pol β is observed and it is mainly distributed inside the nuclear;3. The proliferous speed of EC9706-wtPolβ is significantly slower and the cell number of S period is decrease .It indicates that transfected wtDNA Pol β may be involved in and enhance DNA repair function.