Abstract
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.
Highlights
Atoms of the purine bases, forming 1,2-intrastrand and 1,3-intrastrand cross-links as the major adducts
UTP-specific Incorporation by T7 RNA Polymerase at the Site of a Cisplatin 1,2-Intrastrand d(GpG) Cross-link—We further studied nucleotide incorporation by T7 RNAP at the site of platinum damage under conditions of high NTP concentration, with the aim of determining whether a specific nucleotide might be responsible for this effect
Promoter-dependent and -independent Transcription Inhibition at Platinum Cross-links—The transcription system in which platinum-DNA adducts are immobilized on a solid support provides a powerful tool to investigate the molecular mechanism of transcription inhibition by, and the properties of RNA polymerases stalled at, the major cross-links formed by cisplatin, carboplatin, and oxaliplatin
Summary
Atoms of the purine bases, forming 1,2-intrastrand and 1,3-intrastrand cross-links as the major adducts These DNA adducts inhibit essential cellular processes including transcription and trigger cell death. Several types of DNA lesions, including cisplatin cross-links, inhibit transcription by blocking RNA polymerase (6 –15). The dominant consequence of RNA polymerase blockage by platinum-DNA adducts, either damage repair or apoptosis, will strongly bias the fate of cancer cells treated with the drugs. Different kinds of cisplatin-DNA adducts, 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpNpG) cross-links, provide different levels of transcription inhibition. In vitro transcription by RNA polymerase II in human cell extracts is strongly inhibited by a 1,3-intrastrand but not a 1,2-intrastrand adduct, whereas both types of cross-link efficiently block T7 and SP6 RNA polymerases [10, 19]. The ability of RNA polymerase to resume transcription after being arrested at a platinum-DNA lesion was examined by chemically removing the platinum adduct
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