Mutation and repair of DNA damaged with cisplatin, an anticancer agent

Cisplatin is a potent anticancer drug forming intrastrand-crosslinks (65% GG, 15% AG) in the DNA that are thought to be responsible for its antiproliferative effects. These lesions inhibit replication, cause mutations and lead to secondary damage like daughter strand gaps and double strand breaks. Efficacy of cisplatin in chemotherapy is limited by development of tumor resistance caused by elevated DNA repair leading to damage tolerance.; In this work the roles of Pol II and homologous recombination (HR) in facilitating resistance of Escherichia coli PQ30 and derivatives to cisplatin were studied. Both the RecBCD and RecFOR pathways of HR were important for survival and Pol II was required only in a RecFOR− background after 2 h cisplatin treatment. SOS induction levels were measured by β-galactosidase assays in E. coli strains that have the lacZ gene fused to the sfiA promoter. Early induction of SOS occurred in recB strains whereas induction was delayed in RecFOR− strains.; Effects of mutation in the RecFOR pathway upon cisplatin mediated mutagenesis was studied in E. coli strain CC102 and derivatives. RecFOR mutant strains were severely compromised in survival and the introduction of the recA803 (srf) allele alleviated sensitivity to cisplatin. Frequency of mutation of the rpoB gene (chromosomal) and reversion to Lac+ (episomal G→A) decreased significantly in RecFOR− strains and was restored only to modest levels upon introduction of the recA803 (srf) allele.; Sites where DNA Polymerase stopped in vitro due to cisplatin adducts on the template were determined by primer extension, asymmetric PCR and quantitative PCR assays. In vivo sites of cisplatin induced mutations in the supF gene borne on the plasmid pBB1 were also studied. Survival of plasmid pBB1 and mutation frequency of the supF gene was higher in the SOS induced than in the uninduced E. coli ES87 cells. The in vitro stop sites did not correlate with in vivo sites of mutation in the supF gene except at position 169 (GG site).