REPLICATION INHIBITION AND TRANSLESION SYNTHESIS ON TEMPLATES CONTAINING SITE-SPECIFICALLY PLACED CIS-DIAMMINEDICHLOROPLATINUM(II) DNA ADDUCTS

A series of site-specifically platinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2{d(ApG)-N7(1),-N7(2)}], cis-[Pt-(NH3)2{d(GpCpG)-N7(1),-N7(3)}], and trans-[Pt(NH3)2{d(CpGpCpG)-N3(1),-N7(4)}]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed (cis-[Pt(NH3)2{d-(GpG)-N7(1),-N7(2)}] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2{d(GpCpG)-N7(1),-N7(3)}] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed < 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes. The properties of this adduct in the M13 genome were investigated by postreplication sequence analysis of the translesion synthesis product. Taken together, these studies demonstrate that polymerases can traverse through all of the major bifunctional cisplatin adducts formed in vitro and in vivo and strengthen the hypothesis that adduct-induced mutagenesis may occur through replication bypass.