title: Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent.
abstract: Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.
authors.affiliation: Department of Chemistry and the Whitaker Health Sciences Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: J N
authors.initials: JN
authors.lastname: Burstyn
authors.forename: W J
authors.initials: WJ
authors.lastname: Heiger-Bernays
authors.forename: S M
authors.initials: SM
authors.lastname: Cohen
authors.forename: S J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology*; D018736:DNA Adducts / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D020161:Physical Chromosome Mapping; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D010984:Platinum / Q000378:metabolism; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D016615:Telomere / Q000235:genetics; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D018736:DNA Adducts; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D010984:Platinum; D004259:DNA-Directed DNA Polymerase; D002945:Cisplatin