RESUMO
Structure-specific recognition proteins (SSRPs) bind to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. A yeast gene encoding an SSRP, designated IXR1, was cloned and sequenced. The Ixr1 protein, a member of the high mobility group-box protein family, bound specifically to DNA modified with cisplatin but not inactive platinum compounds. A yeast strain with an inactivated IXR1 gene was half as sensitive to cisplatin and accumulated one-third as many platinum-DNA lesions after treatment with cisplatin as the parental strain. These findings suggest that SSRPs play a role in mediating the cytotoxicity of cisplatin.
Assuntos
Cisplatino/metabolismo , Cisplatino/farmacologia , Adutos de DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA , DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genéticaRESUMO
DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.