突变型DNA聚合酶β碱基切除修复活性的分析

Analysis of base excision repair activity in mutant DNA polymerase β

目的分析162位Val→Ala突变型DNA聚合酶β(DNA polymerase β,polβ)碱基切除修复活性的变化,为探讨修复基因DNA聚合酶β突变在肿瘤发生发展中的作用积累实验依据。方法异丙基-β-D-硫代半乳糖苷(IPTG)分别诱导含有野生型和162位Val→Ala突变型DNA聚合酶β表达载体(pQE80L-Wpolβ和pQE80L-Mpolβ)的大肠杆菌DH5α;通过镍柱亲和层析法纯化蛋白,复性后蛋白定量;退火合成含有单碱基缺失的DNA底物,与纯化的野生型和突变型DNA聚合酶β蛋白进行BER修复试验。结果通过诱导、纯化得到38kd野生型和突变型DNA聚合酶β蛋白;在BER实验中,野生型DNA聚合酶β能够在DNA底物的酶切位点处将其完全切开,而突变型DNA聚合酶β仅部分将其切开。结论162位Val→Ala突变型DNA聚合酶β碱基切除修复能力显著减弱,提示肿瘤发生发展与polβ基因修复活性降低密切相关。

Objective To analyze the variation of base excision repair （BER） activity in mutant DNA polymerase β （polβ） at 162aa （Val→Ala） and provide experimental data for tumor investigation. Methods Isopropyl-β-D-thiogalactoside （IPTG） was used to induce wild type and mutant DNA polymerase β gene which cloned into expression vector pQE80L-Wpolβ and pQE80L-Mpolβ,and transfected into E.coli DH5α.I-IislinkTM spin protein purification system was used to purify Wpolβ and Mpolβ protein.Synthesize the DNA substrate which loss single basic group.Then,the purified protein were used to base excision repair for DNA substrate. Results 38kd wild type and mutant DNA polymerase β protein were obtained through inducement and purification.The BER production of wild type DNA polymerase β can absolutely cut off while the production of mutant DNA polymerase β not. Conclusion The BER activity of the mutant DNA polymerase β at 162aa （Val→Ala） is weaken significantly,it indicate that the development of tumor is correlated to polβ gene.