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Analysis on the Mutated Basic Group Excision Repair Activity of DNA Polymerase β M162

Author: ChenYueQin
Tutor: ZhaoGuoQiang；SongGuoHua
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: DNA polymerase beta Mutation Base excision repair
CLC: R73-3
Type: Master's thesis
Year: 2009
Downloads: 4
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Abstract

The background and purpose of the DNA polymerase β (DNA polymeraseβ, polβ) is an important DNA repair enzyme which is widely present in mammalian cells. The main function of this enzyme is involved in DNA repair in base excision repair (BER) to fill a single nucleotide gap, including short-patch BER pathway (single nucleotide) and / or long-patch (a few nucleotides the Merger) BER way is the today known base excision repair the most important one of the polymerase. Enzyme homology in mammalian cells and in the structural evolution of the height of the conservative description plays an important role in the activities of life, it was called a \The enzyme defect and the occurrence of some tumors. , Found in a variety of human tumor tissues of esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, nasopharyngeal cancer gene mutations polβ and abnormal expression. In recent years, many scholars polβ mutations in tumor tissues and cell lines and expression status of the system. However, mutations in the tumor tissue polβ functional changes have not seen any reports. Accordingly, the subject of the Task Force on the basis of previous studies, build 162 Val → Ala mutant DNA polymerase (DNA polymerase βM162 of mutant) and wild-type DNA polymerase beta expression vector induced expression of purified recombinant beta protein to detect single base deletion ability to repair DNA substrate, in order to investigate the repair gene DNA polymerase beta mutations in tumor development in the accumulation of experimental evidence. Experimental design with BamH I and Hind III restriction sites polβcDNA primer; containing the wild type polβcDNA clone pGEM-T-Wpolβ and 162 Val → Ala mutant DNA polymerase beta (M162 mutant) cDNA clone pGEM- The T-Mpolβ the plasmid as a template for PCR amplification; the amplified by BamH Ⅰ and Hind III digested Wpolβ, and Mpolβ product, which was cloned into pQE80L; using PCR screening and restriction endonuclease to get pQE80L-Wpolβ and pQE80L-Mpolβ DNA sequencing . Respectively 1mmol/LIPTG induced to contain pQE80L-Wpolβ pQE80L-Mpolp of E. coli DH5a, 37 ° C induced culture 5h; sonicated cells, and protein content by UV spectrophotometry. Then 15% SDS-PAGE electrophoresis test Brilliant Blue R-250 staining, methanol - acetic acid bleaching was observed using gel analysis system scans analyzed on SDS-PAGE gel stained strip. Purified protein purified protein by Hislink ^{TM protein purification kit according to the conventional method with 8M urea refolding, the protein content by UV spectrophotometry, the wild-type and mutant DNA polymerase beta protein diluted to 400ng/ml. Designed and synthesized with Hind III restriction sites single base deletion of three single-stranded DNA substrate P1, P2 and P3, with a base deletion annealed synthetic DNA substrate; dilution wild type and mutant DNA polymerase beta protein on the DNA substrate DNA repair substrate base deletion, Hind III-digested DNA repair substrate, it is determined whether the BER activity. Experimental results. Recombinant expression vector pQE80L-Wpolβ the pQE80L-Mpolβ by PCR and restriction enzyme digestion confirmed vector was constructed successfully. DNA sequence analysis confirmed the expression of the inserted fragment and the vector pGEM-T-Wpolβ and sequence in the pGEM-T-Mpolβ completely consistent. 2 through the induction of the expression, 39KD purified wild-type and mutant polβ protein, a fusion protein with the expected size of the same. 3 matches three single chain P1, P2, P3 annealed synthetic 56bp DNA comprising a nucleotide deletion substrate, with the expected size. 4 the BER experiments when diluted 1:4 i.e. polβ100ng/ml, wild type polβBER activity was 100%, while only 29% ± 4% mutant polβBER activity; the 1:8 dilution, i.e. polβ50ng/ml when, wild-type The the the polβBER activity was 78% ± 9%, while only 5% ± 1.3% mutant polβBER activity; 1:16 dilution i.e. polβ25ng/ml is, there are still 32% ± 4% wild type polβBER activity, whereas mutant polβBER activity has been reduced to 0%; statistical analysis, the three dilution wild-type polβBER activity was significantly higher than mutant of pol, p <0.01, the differences are very significant. The conclusions the DNA polymerase βM162 mutation type mutated to C, i.e. DNA 598 T 162 Val cause polβ amino acid mutation as Ala, lead to the the palm space protein polβ activation domain structure change, resulting in BER activity is significantly reduced.}

Analysis on the Mutated Basic Group Excision Repair Activity of DNA Polymerase β M162

Abstract

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