Establishment Of Cell Lines Stably Expressing Containing Wild Type And Mutant DNA Polymerase β Gene And Effection Of Two Type Polymerase β Expression On CHO Cells Growth

Background and objective: Oncogenesis is a consequence of accumulated mutations in multiple genes. The presence of a mutator phenotype facilitates the early stages of tumor growth and progression . This phenotype includes alterations of gene products involved in DNA replication and repair as well as in maintaining genomic stability and integrity. Genes encoding enzymes involved in DNA replication are potential targets for mutations leading to cancer. DNA polymerase β (polβ), a single-copy gene, is involved in base-excision repair (BER) for DNA maintenance. It also may be involved in replication, recombination, and drug resistance in eukaryotic cells .The DNA polp gene, which encodes a 39-kDa protein, consists of 335 amino acids with two distinct functional domains domains of 8 kDa and 31 kDa, separated by a short proteasesensitive region . The N-terminal 8-kDa polypeptide (75 residues) has a strong affinity for single-stranded DNA and is devoid of nucleotidyltransferase activity . The polymerase activity of the polβ enzyme resides in the C-terminal 31-kDa domain ( 260 residues). The 31-kDa domain may bind to a double-stranded DNA. Under normal conditions, polβ_expression levels are low and constant throughout the cell cycle. The replication fidelity of polβ is very low compared with many other DNA polymerases; in in vitro replication assays with pol, the rate of 1 -bpdeletions in mononucleotide runs was higher than the rates of other types of mutations .DNA Polβ gene mutations were observed in ovarian tumors, as well as prostate, bladder, breast, or colon cancer tissues, compared with adjacent normal tissues . In our previous studies, mutations of Polβ have also been detected in human esophageal carcinoma. We found a high occurrence (23%) of mutation as 58bp deletion which encodes amino acid residues 22-40 in binding domain of the enzyme. Futhermore, we constructed eukaryotic expression vectors carrying the wild type and the mutant respectively. Here we establish cell lines expressing containing wild type and the mutant stably and study the effects of the polβ of wild type and the mutant on Chinese hamster ovary cells(CHO) primarily.Methods: The constructed eukaryotic expression vector carrying the wild type and the mutant was transfected into CHO respectively by the method of lipofectamine. The stable transfectants was screened by G418. The expression of mRNA of wild type and the mutant was determined by RT-PCR, Growth curve of the polβ gene transfected CHO cells was drawn with the results of MTT assay. The changes in cell cycle were analyzed by flow cytometry.Result: Cell lines stably expressing wild type and the mutational polβ were established. The mutant can increase the growth rate of CHO cells and the percentage of S phase cells in vitro. The effects of the wild type enzyme on growth rate and cell cycle were not observed.Conclusion: The wild type and the mutant result the different effect on CHO cells. It can be used for the further study of function of the wild type and the mutatantal pol β .