The Role Of TCR In UVC And Cisplatin Induced SupF Gene Mutation In Vivo

Background and objective:1. UV induced DNA damage and repairDNA is certainly one of the key targets for UV and cisplatin-induced damage in a variety of organisms ranging from bacteria to humans.UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane pyrimidine dimmers (CPDs) and 6–4 photoproducts (6–4PPs).However,cells have developed a number of repair or tolerance mechanisms to counteract the DNA damage caused by UV or any other stressors.Excision repair,which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER),plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases,respectively.In addition,mechanisms such as mutagenic repair or dimmer bypass,recombinational repair,cell-cycle checkpoints,apoptosis and certain alternative repair pathways are also operative in various organisms.2. Cisplatin induced DNA damagecis-Diamminedichloroplatinum(II) (cisplatin) is widely used in chemotherapy.The N7 atoms of guanine and adenine are the main binding sites for platinumcom-plexes in double-stranded DNA,The interaction between cisplatin and DNA can result in mono- and bifunctional adducts,as well as DNA–protein crosslinks.The bifunctional adducts,which can take the form of intra- or interstrand crosslinks,may cause major local distortions of DNA structure,involving both bending and unwinding of the double helix.The intrastrand crosslinks cis-Pt(NH3)2-d(GpG) (60–65% of the total) and cis-Pt(NH3)2-d(ApG) (22–30%) are the most abundant products of the interaction.These cross-links,which comprise 90% of the DNA-cisplatin adducts,bend the helix by 34°towards to major groove and unwind it by 13°. 3. The distinction between TCR and GGR pathwayNucleotide excision repair(NER) is divided into two sub-pathways: global genome repair (GGR) and transcription-coupled repair (TCR).NER is a complex but highly conserved process in which basically the following steps can be distin- guished: (i) recognition of a DNA lesion;(ii) single strand incision at both sides of the lesion;(iii) excision of the lesion-containing single stranded DNA fragment;(iv) DNA repair synthesis to replace the excised nucleotides;(v) ligation of the remaining single stranded nick.In eukaryotic cells the process of NER requires more than 30 proteins.The most delicate step in NER is the recognition of the DNA lesions in their different chromatin context and it is not surprising that the mechanism of damage recognition in GGR and TCR is principally different and requires specific proteins.4. The molecular mechanism of TCRTCR first described by Mellon and Hanawaldepends on active RNA poly-merase I and II driven transcription and is specifically targeted to DNA lesions in the transcribed strand of an active gene.DNA lesions that induce poor helix distortion but block transcription elongation are preferentially repaired by TCR resulting in an accelerated repair of DNA lesion in the transcribed strand compared to repair of non-expressed DNA.Lesions that efficiently block transcription may also be efficiently recognized by GGR,due to local disruption of base pairing at the site of the lesion.Thus,the GGR pathway may overrule TCR.It is not clear how repair is coupled to transcription.The favourite hypothesis is that the stalled RNA polymerase is able to attract NER components by direct interaction or to modify local chromatin structure to create optimal conditions for repair.A relevant observation is that the rate repair of structurally different lesions by TCR is very similar indicating that in contrast to GGR,the structure of the lesion is less important for damage recognition in TCR.In any case,it is clear that damage recognition in TCR does not require XPC-hHR23B protein,but depends on other proteins most notably the CSA and CSB proteins.The fact that certain DNA lesions can be repaired without the need of XPC-hHR23B suggests that NER proteins might detect a transcription bubble with a RNA polymerase stalled at a DNA lesion without the requirement of XPC-hHR23B.An alternative view is that for certain lesions (such as those induced by oxidative damage) CS proteins are required to bypass the lesions and hence in the absence of CS the transcription will be persistently inhibited.In this case,the role of CS proteins is to make transcription more processive rather than enhancement of repair.5. We established a controllable transcription system to study the difference of mutation spectra between TCR and GGR pathwayThe Tet-Off and Tet-On Gene Expression Systems give researchers ready access to the regulated,high-level gene expression systems.In the Tet-Off system,gene expression is turned on when Tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture medium.In contrast,expression is turned on in the Tet-On system by the addition of Dox.Both systems permit gene expression to be tightly regulated in response to varying concentrations of Tc or Dox.In our research,the mutation reporter gene SupF was used to construct a special Tet response plasmid pTCR-SupF-Luc,this plasmid was transfected into Tet-on 293 cell line,we could control the transcription of SupF gene in response to varying concentrations of Dox,at the presence of DOX,TCR was the mainly repair pathway,at the absence of DOX,GGR is the mainly repair pathway,so the different mutation rate can be measured in these two pathways.Methods1. The construction and identification of a bidirectional shuttle vector The pBI-L Tet Vector is a response plasmid that can be used to express a gene of interest and Luciferase from a bidirectional Tet-responsive promoter,we obtained SupF gene fragment from pSupF-G1 plasmid DNA,and conjuncted with pBI-L plasmid,so we obtained a subclone plasmid named pTCR-1 it was the first step of gene clone.Then we amplificated SV40ori and Kan/Neo fragment from pEGFP-1 plasmid with PCR method,these fragment were inserted into pTCR-1 plasmid,eventually we obtain a new recombinant plasmid named pTCR-SupF-Luc.It was identificated by restriction enzyme and DNA sequencing.2. To set up a controllable transcription system use Tet-on 293 cell line and the shuttle vector pTCR-SupF-LucThe shuttle vector pTCR-SupF-Luc contains a bidirectional Tet-responsive promoter between the Luciferase gene and SupF reporter gene,and a SV40ori repliacation initiator for eukaryotic cell.This Tet response vector can be transient transfected into Tet-on 293 cell line,and the controllable transcription system was established.The system could be used to research the DAN repair molecular mechanisms.The report genes were turned on in the system via the addition of Dox.At the presence of DOX,TCR was the mainly repair pathway,in contrast,at the absence of DOX,GGR is the mainly repair pathway.Therefore the difference mutation rate can be measured in these two pathways by putting in DOX or not of DOX.3. To study the mutation frequency and mutation spectra of UVC induced TCR pathway in Tet-on 293 cell lineUVC(254 wave length ultra-violet) induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane pyrimidine dimmers (CPDs) and 6–4 photoproducts (6–4PPs).We used UVC as the DNA damage factor to treat pTCR-SupF-Luc plasmid DNA in vitro,then the damaged DNA was transfected into Tet-on 293 cells with liposome,meanwhile the undamaged plasmid were tansfected into Tet-on 293 cells as control.The transfected cells were incubated for 48-hours for DNA repair and replication. The transfected cells were harvested for vector DNA isolation by a modified alkaline lysis procedure. The DNA were transformed E. coli SY204 [lacZ125(Am)]by electroporation. The transformed E. coli cells were plated onto LB plates containing Kanamycin,X-Gal,and IPTG .Mutant colonies containing inactivated SupF genes unable to suppress the amber mutation in the host cellβ-galactosidase gene were detected as white colonies among the wild-type blue ones.The mutant colonies and the total colonies were counted.The mutant colonies were chosen for DNA sequencing.4. To verificate if the Cisplatin mediated DNA crosslinks could induced TCR pathway,and the molecular mechanism of TCR mediated SupF gene mutationCisplatin forms approximately 60-65% intrastrand GG,25-30% intrastrand AG,5-10% intrastrand GNG,and 1-3% interstrand GG diadducts.NER is one of the mainly repair pathway in human cells,but many questions remained unknown that the TCR pathway how to participate the repair process.So we utilized the controllable transcription system to try to address these questions.The pTCR-SupF-Luc plasmid DNA were damaged by cisplatin in vitro,and transfected into Tet-on293 cell line for repair and replication under the condition of DOX (+)and DOX (-).The undamaged plasmid were tansfected into cells as control for spontaneous mutation measurement.After 48 hours,the plasmid DNA were extracted form cells,transformed E.coli SY204.Mutant colonies containing inactivated SupF genes were detected as white colonies among the wild-type blue ones.The mutant colonies and the total colonies were counted.The mutant colonies were chosen for DNA sequencing.Results1.We got a new recombinant plasmid,this plasmid include bidirectional Tet-responsive promoter,two report gene,a SV40 replication origin. DNA sequencing and restriction enzyme digested certificated the correction of insertion fragment.2. We constructed a responsive gene transcription control system,this system included a Tet control cell line,Tet-on293 cell line,and a bidirectional Tet-responsive plasmid pTCR-SupF-Luc.We measured the expression of Luciferase gene at the presence and absence of DOX,when DOX was added into medium,Luciferase expression were significantly rised compare with the absence of DOX,this result demonstrated that the gene transcription control system worked as will,it could be utilized in the investigation of TCR pathway.3. The mutation information induced by UVC as fellow: In TCR pathway,the mutation frequency of SupF gene was equal to 2.2%,the mutation hotpots located in 5'-CG-3',5'-CC-3'and 5'-TC-3'regions;In GGR pathway,the mutation was equal to 1.2%,the mutation hotpots located in 5'-GGGGGGG-3',5'-CG-3'and 5'-TT-3'regions.The point mutations are listed in the following table.4. The mutation information induced by cisplatin as fellow:In TCR pathway,the mutation frequency of SupF gene was equal to 1.45%,the mutation hotpots located in5'-GG-3'and 5'-GCCG-3'regions;In GGR pathway,the mutation was equal to 1. 20%,the mutation hotpots located in 5'-GGGGGGG-3',5'-GGGA-3'and 5'-GAAG-3'regions. The point mutations are listed in the following table. Conclusion:1. we succeeded to construct a recombinant plasmid DNA.2. we have established successfully a Tet-on controlled SupF transport system for transfecting this new recombinant plasmid DNA into Tet-on 293 cell line, which included a pTET-ON plamid.It could control the transcription of SupF mutation reporter gene and luciferase gene.This system could be used to analysis the mutation of SupF gene mediated by UVC and Cisplatin in TCR pathway.3. We confirmed that TCR pathway take part in the repair of UVC and Ciaplatin induced DNA damage in normal human HEK 293 cell.4. TCR is the mainly repair pathway of CPDs(cyclobutane pyrimidine dimers) and cis-1.3-d(GTG) intrastrand crosslinks.5. We firstly definited the mutation spectra signature of SupF reporter gene in TCR and GGR pathway in tet-on 293 cell line.6. The difference of mutation frequency and spectra between TCR and GGR induced by cisplatin and UVC maybe suggest that the TCR and GGR pathway played different but complementary rols on the maintaining of genetic stability.