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The Role of TCR in UVC and Cisplatin Induced SupF Gene Mutation in Vivo

Author: LiJin
Tutor: SongBo
School: Third Military Medical University
Course: Surgery
Keywords: Nucleotide excision repair (NER) Pan - genome repair (GGR) Nucleotide excision repair (TCR), UVC Cisplatin SupF reporter gene Luciferase reporter gene Tet-on gene expression control system Targeted Mutagenesis studies Mutation frequency Mutation spectrum
CLC: R346
Type: PhD thesis
Year: 2008
Downloads: 63
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Abstract

Research background and research purposes. Different mechanisms of damage identification is the main difference between the transcription-coupled repair and pan-genome repair pathway nucleotide excision repair pathway (NER) is the most flexible one DNA repair pathway, it can repair multiple discontinuous structure of DNA damage. The system need to include XPA, XPB, XPC, XPD, XPF, XPG and ERCC1, including at least 17 species of the vitality of the enzyme or protein. In NER, the rate-limiting step of DNA damage recognition / excision. NER can be divided into two sub-pathways: (1) pan-genome repair (Global genome repair, GGR). The ② transcription-coupled repair (Transcription coupled repair TCR) of. These two approaches differ in the start portion of GGR acting on the non-transcribed region of the DNA, TCR acting on the activity of the transcribed region, and once started, use of the two enzymes. If the injury occurs in the non-transcribed strand of DNA, the occurrence of the pan-genomic repair, formation of XPC/hHR23B complexes is the initial stage of the NER damage recognition. If the injury occurred on the transcribed strand of active genes parts transcription coupled repair occurs role as a transcription of RNA polymerase II is blocked, lead to rapid TCR start signal. In this process, some wrong with protein in the repair process CSA and CSB (CS, Cockayne syndrome, Kane syndrome) is required, CSA and CSB to be the stagnation of RNA polymerase II to move function to allow there is enough space to complete the NER. This repair is rapidly than GGR and higher efficiency. Cisplatin and UV-mediated DNA damage theoretically can occur in many exogenous DNA damage factors mediated TCR, most research cisplatin (cisplatin) and ultraviolet radiation (UV). Cisplatin add composition (Pt-DNA) and the DNA cross-linking (DNA-crosslink) and damage DNA chain is formed, to prevent DNA replication, wherein 1-2 chain crosslinking more than 90%, a few 1-3 chain crosslinked. Ultraviolet radiation causes cyclobutane pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs) and 6-4 photoproducts formation damage DNA. UV radiation-induced DNA damage is the fundamental reason that it can induce adjacent pyrimidine bases in the same strand of the DNA pyrimidine dimer DNA spatial structure changes, thus preventing DNA replication, transcription and thus affect the biological function of the protein. Cisplatin and UV-mediated DNA damage in common is that the cross-linking between the DNA chain or chain can lead to spatial changes in the structure of the DNA double helix, hinder DNA transcription, in theory, can be mediated transcription-coupled repair pathway place. 3. Cisplatin and UV-mediated TCR and the relationship of the mutation occurs in vitro studies in the absence of normal cells within the existing experimental evidence showed UV resulting the CPDs injury can be expressed gene transcription chain repair than in the non-transcribed strand much more rapidly. Instead, the light compounds 6-4 in the expression sequence and a non-expressed sequence product can be fully cleared; cisplatin lead 1-2 chain crosslinking occur in vitro also induce a TCR in a human cell The nuclear lysate ,1-3 cross-chain chain also can block transcription of RNAP Ⅱ. UV and cisplatin DNA damage cells deficient cells tools GGR deficient cells XPA, XPC, the TCR-deficient cells CSA, CSB deficient cells GGR or TCR ways deletion in the characteristics can be individually the molecular mechanism of a certain kind of repair pathways, but the function of the DNA repair deficient cells overall sound, relative to normal cells, does not truly reflect the physiological state of cells to repair and mutations occur. The GGR pathway in the same cells and TCR pathway alone to study the TCR and mutations in the molecular mechanisms of key issues in the normal cells. Various repair pathways in HEK293 cells were normal after DNA damage by the body's various internal and external factors, the cells will start TCR and GGR including, the various ways to repair mutations produce the result of the combined effects of the various repair pathways . In order to achieve with an intracellular GGR and TCR pathway, acting alone, the need to design a controlled reporter gene transcription model, achieved by artificially controlling the transcription of the reporter gene transcription-coupled repair pathway startup, so as to achieve transcription coupled repair and separation of the pan-genome repair. 5 The experimental design ideas and research purposes (1) mutation study the design of the experimental model of tetracycline is regulated gene expression system, known as the Tet-on gene expression system. Theoretically zero, the level of target gene expression in the absence of tetracycline and in the desired gene is efficiently expressed in the addition of a tetracycline derivative doxycycline (doxycycline, Dox). The SupF gene encoding tRNAs can recognize the amber mutations in the lacZ gene of the host bacteria, generating the normal β-galactosidase, containing the β-galactosidase substrate X-gal and the lac operon inducer IPTG and the corresponding Resistance LB indicator plates, blue colonies in rendering. When SupF gene mutations affect or complete loss of the function of its encoded tRNA, the colonies appear tinged with blue or white. In this experiment, we will Tet-on gene expression regulation system and SupF tRNA transit systems are combined to establish a controlled transcription system. To damage the target gene to SupF UVC and cisplatin in vitro injury into the Tet-on293 cells repair and amplification, and finally transformed bacteria picked up the white mutant clones, the mutant DNA sequencing analysis to determine the molecular mechanisms of mutagenesis and cold hotspot mutations occur. ② This study was designed comprehensive studies of transcription-coupled repair progress, the main topic around the transcription-coupled repair mechanism of cisplatin and UV-induced DNA damage and mutation process research proposed by the recombinant plasmid dye, calibration mutation research, blue-white screening, DNA sequencing technology, at the molecular level to explore the following issues: GGR and TCR pathways in the same cell, how to achieve alone; ② The UV and cisplatin induced DNA damage whether induction of transcription-coupled repair pathways occur; report the characteristics of the gene mutation spectrum ③ UV and cisplatin-mediated transcription-coupled repair process; possible molecular mechanism of the ④ transcription-coupled repair pathway mediated mutation occurs; methods: 1. bidirectional promoter , bis shuttle plasmids pTCR-SupF-Luc reporter gene Construction and identification of the Tet-on reaction of plasmid pBI-L as a carrier, insertion mutant reporter gene SupF fragment derived from the plasmid pSupF-G1, and then design for a primer from the plasmid pEGFP- amplification of the SV40 ori / Kan / Neo fragment, after suitable digestion process, and has been subcloned plasmid connection, and ultimately get the purpose plasmid pTCR-SupF-Luc, and by means of restriction endonuclease and sequence analysis to verify insert the position of the sequence, and the correctness of the base sequence, for further studies to provide a sensitive and reliable experimental tools. The establishment and functions of the transport system in the Tet-on control SupF tRNA verify the complete control of the Tet system of plasmids and Tet response plasmid regulation by Tet Tet-on293 cells is stable transfected Tet regulating plasmid pTET-ON plasmid HEK293 The cells, in this experiment as a regulating system that has been constructed and sequenced verified pTCR-SupF-Luc for the reaction system. Cultured the Tet-on293 cells, cationic liposome transfection method pTCR-SupF-Luc cells transiently transfected with the Tet-on293, and 8 h after the replacement of complete medium was added to a final concentration of 1μg/ml doxycycline (DOX) induced transcription detection Luciferase gene expression, 48h later to verify transcription can be carried out. Cells were lysed, and the plasmid was extracted, purified and transformed into special bacteria a SY204 authentication SupF reporter gene activity. UVC-mediated transcription-coupled repair and mutation occurred molecular mechanism of UVC (wavelength of 254nm UVC) in vitro injury pTCR-SupF-Luc plasmid dose 1500J/M2, plasmid cationic liposome injury stained the Tet-on293 cells, cells were divided into dosing group and the control group, to replace the complete medium 8h after dosing group was added to a final concentration of 1μg/ml DOX cells in each group continued to 48h, the plasmid can be fully replication and repair. A portion of cells after lysis measured Luciferase gene expression, mutation significantly higher SupF reporter gene transcription start cracking the remaining cells, extracted plasmid transformed SY204 bacteria, white mutant clones were picked up by the blue-white screening, calculated mutation frequency = The number of white colonies / total number of colonies. Amplified and sequenced for mutation spectrum, respectively, compared transcriptional and non-transcriptional state of mutation, analysis of the molecular mechanisms of mutation in the UVC-mediated transcription-coupled repair process. 4. TCR in the mechanism of action of the cisplatin led the SupF genetic damage and mutations occur the study pTCR-SupF-Luc plasmid 100μg, was added to a final concentration of 50uM cisplatin solution to a total volume of 200 μl at room temperature for 4h plasmid fully exposed the formation of DNA cross-linking. Phenol: chloroform extraction and ethanol precipitation of purified plasmid, cationic liposomes transfected the Tet-on293 cells targeted Mutagenesis studies, the mutant clones to determine the mutation frequency and DNA sequencing were picked up by the blue-white screening of bacteria, mutation characteristics were measured transcriptional and non-transcriptional state SupF analysis of transcription-coupled repair mechanism of cisplatin mutagenic process. Results 1. Successfully build a bidirectional promoter dual reporting the shuttle plasmid genes pTCR-SupF-Luc, restriction enzyme digestion and sequencing to determine the location of the inserted fragments meet the design requirements, 100% of the base sequence and source sequence homology. 2 cells with Tet-on293 pTCR-SupF-Luc plasmid combination Tet-on control SupF tRNA transport system controllability and sensitivity of the system after the Luciferase gene the detection and SupF gene activity was measured to prove this system has the following characteristics: ① The SupF mutant reporter gene transcription through the Tet-on system precise regulation; ② The dual-reporter gene: the Luciferase gene SupF gene, through the detection Luciferase gene's expression can clear SupF genes whether to be transcribed, and then clear the whether induced TCR pathway; ③ shuttle plasmid PtCr-supF-Luc plasmid containing the prokaryotic cell replicon Col E1 and eukaryotic cell replication SV40ori survival and replication in bacteria and mammalian cells, such that occur in mammalian cells. genetics events, can be easily obtained in bacteria reported; ④ the SupF gene is small, clear genetic background mutation hot spots and on the downstream sequence becomes relatively easy to locate by DNA sequencing. 3 obtained SupF mutations of the mutation frequency in different transcriptional state reporter gene induced by UVC and mutant spectrum. TCR pathways, SupF gene induced transcription, the mutation frequency is 2.2%, mutation hotspots concentrated in the 5'-CG-3 ', 5'-CC-3', 5'-TC-3 'region the; of GGR way supF gene transcriptional silencing , the mutation frequency is 1.2% mutation hot spots focused on the 5'-ggggggg-3 ', 5'-CC-3', 5'-TT-3 'region. Mutation spectrum is shown in the table below. SupF gene in the cisplatin-induced mutation frequency and different transcriptional state mutation spectrum, TCR pathways, supF gene induced transcription, mutation frequency was 1.45%, mutation hotspots concentrated in the 5'-CG-3 ', 5'- GCCG-3 'region; of GGR pathway, SupF gene transcriptional silencing, the mutation frequency of 1.20%, mutation hotspots concentrated in the 5'-ggggggg-3', 5'-GGGA-3 ', 5'-GAAG-3' region. Mutation spectrum is shown in the table below. Conclusion 1. Successfully constructed the bidirectional transcription dual reporter gene for transcription-coupled repair research Tet-on the response plasmid pTCR-SupF-Luc. 2 In this study, the use of the composition of the the Tet-on293 cells and recombinant plasmid pTCR-SupF-Luc controlled SupF report gene-targeted mutation system functional verification in line with theoretical expectations, the molecular mechanism for the study of transcription-coupled repair and mutation provide an ideal experimental model. Reported transcription coupled repair involved in the UVC and cisplatin-mediated DNA damage repair process in normal human cells. UVC-mediated the CPDs injury (cyclobutane pyrimidine dimers) and cisplatin led cis-1.3-d (GTG) chain crosslinking by TCR priority repair. 5 the first time in the SupF gene to determine the mutation spectrum characteristic differences in the state of normal cells TCR and GGR two repair. Found through analysis of the mutation spectrum in the UV and cisplatin-mediated DNA damage repair process, TCR and GGR has a different mutation fingerprint \kinds of NER repair pathway may play different but complementary role to maintaining the genetic stability of the genome.

The Role of TCR in UVC and Cisplatin Induced SupF Gene Mutation in Vivo

Abstract

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