Analysis On The Mutated Basic Group Excision Repair Activity Of Dna Polymerase β M162

Background and ObjectiveDNA polymeraseβ, as one of the most important DNA repair enzymes, commonly exists in mammalian cells. Its primary function is to involve in DNA repair during the procession of basic group excision repair (BER) make up the shortage of mononucleotide, including single-nucleotide and multi-nucleotide (the combination of several nucleotides), therefore it is the most important known polymerase in BER currently. The enzyme's homology in mammalian cells and the high stability in structural evolution have proved its significance in life activities, thus it is called "basic enzyme". Whats more, the deficiency of this enzyme is related to the causing of some tumor diseases. Up to now, the mutation and the abnormal expression of polβhas been found in many human tumor tissues, such as carcinoma of rectum, esophageal carcinoma, gastric cancer, nasopharyngeal carcinoma, etc.In recent years, researchers have accompolished many systematic researches on the mutation and the abnormal expression of polβin tumor tissues. However, None report on the function alternation of polβin tumor tissues has provided. Hence, based on the former researches, this project is to construct models of the 162 bit Valâ†'Ala mutated DNA polymeraseβM162 and the wild type DNA polymerase B expression vector; to induce the expression, to purify to obtain protein; to test its repair ability to the signal basic group-absent DNA substrate; and to accumulate trail statistics in order to exploration the funtion of the accruing and developing of repair DNA polymeraseβmutation in affecting tumor.MethodsTo design a couple of DNA primers with BamH I and Hind III enzyme site; and take the two plasmids, the one with the wild polβcDNA cloned pGEM-T-Wpolβ, the other with the 162 site Valâ†'Ala mutated cDNA polymeraseβM162 cloned pGEM-T-Mpolβ, as the template respectively to achieve the PCR amplification. Recombinate the amplification production of the BamH I and HindIII Wpolβand Mpolβinto pQE80L to obtain pQE80L-Wpolβand pQE80L-Mpolβthrough PCR filtration and enzyme site measurement, then process the DNA sequencing. After induced the DH5αescherichia coli respectively with pQE80L-Wpolβand pQE80L-Mpolβ5 hours under the temperature of 37℃, break the cells by using ultrasonic, then test the protein level by ultraviolet spectrophotometry. After the follow process of 15%SDS-PAGE electrophoresis, Coomassie brilliant blue R-250 dyeing, Colonial spirit-glacial acetic acid decolorizaton observation, and the gelatum analytical system scan, analysis the dyeing strip on the SDS-PAGE gelatum. To depurate the protein through Hislink^TM protein depurating kit, renaturate the depurated protein through 8M urea renaturation, then test the content of the renaturated protein through ultraviolet spectrophotometry, after that dilute wild DNA polβand mutated DNA polβseparately as 400ng/ml. To design and synthesize three single strands (P1, P2, P3), which contents single basic group missing substrate on HindIII enzyme site, then form one basic group missing substrate after renaturation, use the obtained two dilutions to repair the basic group missing substrates, thus to observe the BER repair activity.Results1 Restore the expression vectors of pQE80L-Wpolβand pQE80L-Mpolβ, after the PCR and the appraisal of Hind III enzyme site, to testify the accomplishment of the vector construction. Through DNA sequencing assessment, to prove the identical sequence of the inserted element of the vector construction and of the pGEM-T- Wpolβand pGEM-T-Mpolβ.2. After inducement and depuration, get 39kd pGEM-T-Wpolβand pGEM-T-Mpolβ, which is with the equal anticipated protein content.3. Synthesize the renaturated three single strand of P1,P2,P3, to get one 56bp basic group missing substrate, which matched the anticipation.4. In BER, when in 1:4 diluting, i.e. polβ100ng/ml, the excision repair activity of pGEM-T-Wpolβis 100%, while the excision repair activity of pGEM-T-Mpolβis 29%±4%; when in 1:8 diluting, i.e. polβ50ng/ml,the excision repair activity of pGEM-T-Wpolβis 78%±9%, while the excision repair activity of pGEM-T-Mpolβis 5%±1.3%; when in 1:16 diluting, i.e. polβ25ng/ml,the excision repair activity of pGEM-T-Wpolβis 32%±4%, while the excision repair activity of pGEM-T-Mpolβdecreased to 0%. The statistical analysis show that, on the three dilute strengths, the BER repair activity of pGEM-T-Wpolβwere all notably higher than that of pGEM-T-Mpolβ, p<0.01, with a high disparity.ConclusionThe mutated DNA polymeraseβM162, DNA 598nt T mutated into C, conduced 162 site Val of the polβamino acid mutated into Ala, then conduced the change on alma spatial structure of polβprotein structural domain, at last reduce the BER activity severely.