Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents

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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)(2)Pt}(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.
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date
2003-12
Language
English
Article Type
Article
Keywords

INTRINSIC TRANSCRIPT CLEAVAGE; NUCLEOTIDE EXCISION-REPAIR; IN-VIVO; ELONGATION COMPLEXES; TEMPLATE DNA; CISPLATIN ADDUCTS; II TRANSCRIPTION; THYMINE DIMER; CROSS-LINKS; BINDING

Citation

JOURNAL OF BIOLOGICAL CHEMISTRY, v.278, no.52, pp.52084 - 52092

ISSN
0021-9258
DOI
10.1074/jbc.M310120200
URI
http://hdl.handle.net/10203/173658
Appears in Collection
CH-Journal Papers(저널논문)
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