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Template primer inactivation by cis- and trans-dichlorodiammine platinum for human DNA polymerase alpha, beta, and Rauscher murine leukemia virus reverse transcriptase, as a mechanism of cytotoxicity.
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1976
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Ss DnaGeneticsDna AnalysisMolecular BiologyNucleic Acid BiomarkersPlatinated TemplatesNucleic Acid ChemistryMolecular DiagnosticsRadiation OncologyTrans-dichlorodiammine PlatinumBiochemistryOligonucleotideDna ReplicationGene ExpressionChromatinChromatin StructureNatural SciencesNucleic Acid BiochemistryNucleic Acid AmplificationTemplate Primer InactivationMedicineCis IsomerMutagenesis
Activated salmon sperm DNA (SS DNA) and poly[d(A-T)·d(A-T)] were incubated with various concentrations of cis -dichlorodiammine platinum [ cis -Pt(II)] and other platinum compounds. Following removal of unbound platinum and the determination of the ratio of bound platinum per base nucleotide ( rb ), the template primers were assayed for template activity using partially purified human DNA polymerase α and β from human lymphocyte cell lines RPMI 1788 or RPMI 8402 and Rauscher murine leukemia virus reverse transcriptase. For cis -Pt(II)-treated SS DNA, the amount of bound platinum per base nucleotide required to inhibit template activity by 50% ( rb -50) ranged from 0.009 to 0.015 (for the three DNA polymerases) while for SS DNA treated with the inactive trans isomer, the rb -50 values were higher by a factor of only 2 (0.02 to 0.026). Therefore, the basis for antitumor activity residing only in the cis isomer is not due to the frequency of nonspecific reactions with DNA. However with poly[d(A-T)·d(A-T)], the rb -50 values for cis -Pt(II) (0.006 to 0.008) were five to seven times lower than those for trans -dichlorodiammine platinum II, suggesting that the basis of selectivity may reside in the ability of the cis isomer to react in a specific, molecularly defined configuration that would be difficult for the trans isomer to form. When SS DNA was treated with platinum-uracil blue or platinum-thymine blue, two second-generation platinum compounds, the rb -50 values were about one order of magnitude higher than those obtained with cis -Pt(II). While preincubation of DNA polymerase α with platinated DNA inhibited the incorporation rate upon being subsequently assayed with an untreated DNA, preincubation of DNA polymerase β with platinated templates enhanced the incorporation rate. In contrast, preincubation of DNA polymerase α with cis -Pt(II) alone for up to 2 hr, inhibited the enzymatic activity only at very high platinum concentrations. It was concluded that only reactions of cis -Pt(II) with template contribute significantly to the inhibition of DNA synthesis.