Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents.
Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used to study stalled transcription elongation complexes. In vitro transcription studies were performed in both a promoter-dependent and -independent manner. An elongation complex is strongly blocked by cisplatin 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpTpG) cross-links located on the template strand. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion, the nature of which can be altered by the choice and concentration of NTPs. The [(1R,2R-diaminocyclohexane)Pt]2+ DNA adducts formed by oxaliplatin, which carries a stereochemically more demanding spectator ligand than the ammine groups in cisplatin, also strongly block the polymerase with measurable differences compared with cis-[(NH3)2Pt]2+ lesions. Elongation complexes stopped at sites of platinum damage were isolated and characterized. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. We also discovered that a polymerase stalled at the platinum-DNA lesion can resume transcription after the platinum adduct is chemically removed from the template.
Jung
Yongwon
Y
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Lippard
Stephen J
SJ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000494:pharmacology*; D018899:CpG Islands; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003486:Cyanides / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D007477:Ions; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009944:Organoplatinum Compounds / Q000494:pharmacology; D000077150:Oxaliplatin; D010984:Platinum / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014544:Uridine Triphosphate / Q000737:chemistry; D014764:Viral Proteins
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D003486:Cyanides; D007477:Ions; D009944:Organoplatinum Compounds; D014764:Viral Proteins; D000077150:Oxaliplatin; D010984:Platinum; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D002945:Cisplatin; D014544:Uridine Triphosphate