The Function Of Translesion DNA Synthesis Of DNA Polymerase Iota And Its Splicing Variant

Background and objective:Cellular DNA encounter various types of DNA damage, include endogenous factors and exogeneous factors. The endogenous factor contain misreplication, random base damage(depurinations,deaminations) ; Byproducts of metabolism(base oxidation, base methylation, base hydrolysis , base mismatch).The exogenous factors contain chemistry(chemical mutagen);physics(ultraviolet ray,γray). The UV light is electromagnetic radiation emitted from the sun (or an artificial source), invisible to the human eye. UV radiation is divided to three areas according to its wavelength: UVA 315–380 nm,UVB 280–315 nm, and UVC 190–280 nm . The energy content of the radiation is inversely correlated to its wavelength, rendering UVC the most harmful component of UV light. The ozone layer absorbs solar UVC and most of UVB, and it is estimated that 1–10% of UV radiation on the surface of Earth is UVB and over 90% UVA. However, the proportion of shorter wavelengths is increasing due to stratospheric ozone depletion. UV radiation has many effects on skin, including inflammation, immunosuppression, and alterations in the extracellular matrix (ECM), in addition to accelerated skin aging. UV radiation also harms the eyes, especially by promoting age-related ocular diseases. The most hazardous effect of excess UV light for humans is, however, increased risk of skin cancers. Cells contain photosensitive molecules, chromophores,which, upon receiving photons in UV radiation, subsequently lift electrons to a higher energy state. A chromophore may pass its excited energy to another molecule and cause a chain reaction. As the occurrence and nature of these events depend on the chemical structure of the chromophore, wavelength of radiation, and specific reaction conditions, the complexity and amount of different biomolecules result in multiple physical events and alterations in cellular functions The main cellular chromophores for UV radiation are DNA and reactive oxygen-generating chromophores. Due to the aromatic ring structures of its bases, DNA absorbs shortwavelength UV very efficiently and is the main chromophore for UVC, but absorbs also a significant amount of energy from UVB. The most apparent types of UV radiation-induced DNA damage are cyclobutane-type pyrimidine dimers (CPDs) and (6-4)-photoproducts (6-4PPs), which cross-link adjacent DNA bases. These UV induced distortions in the DNA helix halt RNA polymerase (RNAP) elongation along DNA, thus inhibiting gene expression. In addition, the active repression of transcription initiation occurs by phosphorylation of RNAPII. The helix-distorting DNA adducts induced by UV radiation, certain chemicals, and oxidative damage are repaired by nucleotide excision repair (NER) in mammalian cells.if the nucleotide excision repair is deficient or there is not enough time to repair all the errors ,there were some unrepaired DNA damages which will interrupt the basic cell process ,for example transcription,DNA replication,maybe lead to gene mutation or cell death.Because the classical DNA polymerase can not bear the twist spatial structure caused by DNA damage,the classical DNA polymerase can not go through the damaged DNA.How to deal with the remain damaged DNA? Y-family DNA polymerases are found in recent years,Y-family DNA polymerases are widely distributed among the three kingdoms of life. Human cells contain at least four; Rev1, polКand two RAD30 paralogs, polιand polη. The Y-family polymerases replicate DNA in a distributive manner and lack any intrinsic exonucleolytic activity for proofreading. In addition to bypassing damaged template or bulky DNA adducts, these polymerases replicate undamaged DNA with a 10- to 100-fold increased error rate compared to the A-, B-, Cand X-family polymerases. Polηis unique among eukaryotic DNA polymerases in its proficient ability to replicate through a cis–syn thymine–thymine (TT) dimer. Remarkably, Polηreplicates through lesion with the same efficiency and accuracy with which it replicates through undamaged Ts, and steady-state kinetic studies have shown that both yeast and human Polηinsert an Aopposite the 3-T and the 5-T of the TT dimer with the same efficiency and accuracy with which they insert an A opposit e a T in the undamaged sequence . Polηhas high fidelity ,because Polηhave 3′to 5′exonuclease activity . DNA polymeraseιis a member of the Y family of DNA polymerases, because polιdefect 3′to 5′exonuclease activity, the fidelity of polιis very low. The dramatic proneness to skin cancer of XP-V individuals,who are defective in DNA polymeraseηtestifies to the importance of this DNA polymerase in cancer avoidance. Indeed, mutations in the XPV gene that cause severe truncations of the protein or prevent the enzyme from relocalising into replication foci after UV radiation result in the xeroderma pigmentosum variant syndrome, one symptom of which is a strong susceptibility to sunlight-induced skin cancers.so we suppose the cells which defect Polηare irradiated by UV ,polιrepair the damaged DNA caused by UV.because of the low fidelity of polι,the rate of mutations is increased ,at last the cancer is formed. For conforming this question,we construct the HEK-293 cells of over-expressed polι.Yang J, et al found a Polι's homologous isomer which is shorted of the fifth exon of Polιcompaired with Polι, and named it PolιS5, then, they cloned the Polιand PolιS5 gene and build their eukaryotic expression vector, but didn't study the specific function of PolιS5. Recent analyses of sequence and microarray data have suggested that alternative splicing plays a major role in the generation of proteomic and functional diversity in metazoan. organisms. Different splice variants of a given protein can display different and even antagonistic biological functions. Efforts are now being directed at establishing the full repertoire of functionally relevant transcript variants generated by alternative splicing, thespecific roles of such variants in normal and disease physiology, we want to know more about the function of polιs5,so we constructed HEK-293 cells of over-expressed polιs5.Methods:1,Stably overexpression of Polιand PolιS5 in HEK293 cells.Pcdna3.1+-Polιand Pcdna3.1+-PolιS5 is saved by our laboratory . Firstly, Pcdna3.1+-Polιand Pcdna3.1+-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).We import the eukaryotic expression vector PCDNA3.1 which contain our object geneiota or iotas5 into the HEK-293 cells with liposome and select the resistance clone by G418.detect the stable over-exspression clone by rt-PCR.2,Cellular localization of Polιand PolιS5 in cells. In order to improve our understanding of the biological function of Polιand PolιS5, we have analysed its cellular localization. The cDNA encoding enhanced green fluorescent protein(EGFP) was fused in-frame to the N-terminus of Polι(EGFP-C1-Polι) and PolιS5 (EGFP-C1-PolιS5), and then pEGFP-C1 vector, pEGFP-C1-Polιand pEGFP-C1-PolιS5 were transfected into HEK293 cell respectively. Due to the nuclear localization signal consensus motif in its amino acid sequence, EGFP were localized within the nucleus and appeared homogeneously distributed. The distribution of EGFP-Polιand EGFP-PolιS5 in the nuclear localization were detected by by Laser scanning confocal microscope. We irradiated the transfected cells with UVC(15 J/m2) and observed change of distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 in the nuclear localization 4 h later.3,Compare the UV sensitivity of HEK-293 cells,iota- HEK-293 cells and iotas5- HEK-293 cellsHEK-293 cells,pCDNA3.1+-iota HEK-293 cells and pCDNA3.1+-iotas5 HEK-293 cells digested by pancreatinum zymine were plated in 96 pore plates, 8000 cells every pore.The second day ,we use different dose of UV to irradiat the cells .after 24 hours or 72 hours ,we use the method of MTT to detect the survival rate .4,Determine the function of polιand polιs5 in UV-induced mutagenesisRadiate the SUPF with UV(3000J/m2),then transfection the radiated supF into HEK-293 cells,over-expressed iota HEK-293 cells,over-expressed iotas5 HEK-293 cells.After forty-eight hours,we extracted the supF from these cells and used DPNⅠto digest for three hours,then transformed the digested supF into SY204 bacteria,planked the bacterias on the plates which contained X-gal,IPTG and ampicillin, the seconed day ,there are a lot of blue spots and a few of white spots on the plates. We count the amount of spots to get the frequency of mutations.we picked the white spots and sented it to the shanghai shenggong company for getting the sequence of the supF gene.Results:1,The polιor polιs5 was stably overexpressed in HEK293 cells. Pcdna3.1+-Polι,Pcdna3.1+-PolιS5 were transformed into Escherichia coli DH5αand were mini-extracted, then they were sequenced and identified by Blat (www.genome.ucsc.edu).It was determined that the cDNA of PolιS5 in Pcdna3.1+PolιS5 is shorted of the fifth exon of Polιcompaired with the cDNA of Polιin Pcdna3.1+-Polι. The next experience will be based on the result.We extract the total RNA,we found the band of 18s and 28s is very clear by electrophoresis.the brightness of 28s is two times more than the brightness of 18s,the brightness of 5s is gloomy.we selected clones witch can resistant G418,then we detected the stable over-exspression clone by rt-PCR.2,The cellular localization of Polιand PolιS5 in cells were detected.EGFP were localized within the nucleus and appeared homogeneously distributed. The EGFP-C1-Polιand EGFP-C1-PolιS5 were predominantly localized within the nucleus and appeared homogeneously distributed. In 2% of EGFP-C1-Polιtransfected cells, and in 4% of EGFP-C1-PolιS5 transfected cells, EGFP- C1-Polιand EGFP-C1-PolιS5 were localized in many intranuclear foci. This localization pattern was strikingly similar to observations with pol eta .Consequently, we asked whether the distribution of EGFP-C1-Polιand EGFP-C1-PolιS5 changes after DNA damage. We irradiated the transfected cells with UVC(15 J/m2) and observed the pattern of EGFP-C1-Polιand EGFP-C1-PolιS5 4 h later by Laser scanning confocal microscope. It were suggested that PolιS5 probably has the similar function with Polιin translesion sythesis.3,The UV sensitivity of HEK-293 cells,iota- HEK-293 cells and iotas5- HEK-293 cells were measured with MTT assay.We used different dose of UV to irradisted the cells.After 24 hours ,we found the survival rate of HEK-293 cells were from 100% to 72%, The survival rate of Pcdna3.1+-PolιHEK-293 cells were from 100% to 72%, The survival rate of Pcdna3.1+-Polιs5 HEK-293 cells were from 100% to 82%,they had no difference. We used different dose of UV to irradisted the cells.After 72 hours ,we found the survival rate of HEK-293 cells were from 100% to 16%, The survival rate of Pcdna3.1+-PolιHEK-293 cells were from 100% to 12%, The survival rate of Pcdna3.1+-Polιs5 HEK-293 cells were from 100% to 14%,they had no difference They had no difference too.4,The function of Polιand PolιS5 in UV-induced mutagnesis were investigated with the in vivo supF mutation detection system. we successfully transfected the supF into HEK-293 cells,over-expressed iota HEK-293 cells and over-expressed iotas5 cells,then extracted the supF from these cells,and efficiently transformed it into SY204 bacterias。We get the frequency of mutations in different cells and the mutation spectrum by sequencing.Conclusion:1. polιand polιs5 accumulate to UV lesion site and respond to the UV damage in vivo,2. polιand polιs5 didn't effect the UV sensitivity of HEK293 cells.3. polιand polιs5 contribute to the UV induced mutagenesis.4. PolιS5 probably share the similar function with Polιin translesion sythesis.