Base-pair substitution hotspots in GAG and GCG nucleotide sequences in Escherichia coli K-12 induced by cis-diamminedichloroplatinum (II).

Abstract

Cell killing and mutation induction by cis-and trans-Pt(NH3)2Cl2 in Escherichia coli were examined by studying forward mutagenesis in the lad gene in cells with different repair capacities. Survival experiments showed that repair-proficient cells were slightly more sensitive for the cis isomer than for the trans isomer, whereas repair-deficient RecA and UvrB cells were extremely sensitive only for the cis compound. cis-Pt(NH3)2CI2 induced mutagenesis in both wild-type cells and RecA cells but not in UvrB cells; whereas no detectable mutagenesis was induced by treatment with the trans compound. Examination of the nature of the mutations induced by cis-Pt(NH3)WCI2, by. using the Lad system, revealed that base-pair substitutions leading to nonsense mutants are only induced in wild-type cells, suggesting that the intact products of both the uvrB and the recA gene are necessary for the repair responsible for this type of mutagenesis. Investigation ofthe nonsense mutants reveals that 70% ofthese mutations result from GC --TA or GC --AT substitutions at sites where the guanine is part of a GAG or GCG sequence. These results are discussed in relation to existing theories on the interaction between Pt compounds and DNA. A model for Pt-DNA adducts, leading to base-pair.substitutions, is proposed.About a decade ago, Rosenberg et aL (1) reported that cisPt(NH3)2CI2 shows antitumor activity against sarcoma 180 and leukemia L1210, whereas trans-Pt(NH3)2C12 is ineffective.Since then it has been shown that several other cis Pt(II) and cis Pt(IV) compounds exhibit antitumor activity (2, 3). In mammalian, as well as in bacterial cells, DNA is the preferential target for Pt compounds. For cis-Pt(NH3)2CI2 this interaction results in lesions that selectively block DNA replication (4, 5). In this respect, cis-Pt compounds behave similar to other drugs such as alkylating and radiomimetic agents.In vitro, cis-Pt(NH3)2C12 binds to bases in DNA, and the order of binding affinity has been shown to be guanine>ad-enine>cytosine>>thymine, with a strong preference for the N-7 position of guanine (6). Monofunctional binding to a single base is unlikely to be the principal lesion through which cisPt(NH3)2CI2 exerts its antitumor activity because, at equitoxic doses, more of the inactive trans compound is bound to DNA (7). Therefore, specific bifunctional binding of cis-Pt(NH3)2CI2 to DNA is thought to be responsible for its antitumor activity.For the bifunctional mode of action, several models have been proposed such as intrabase chelation at the 0-6 and N-7 positions of guanine (6,8), interstrand crosslinking between the N-7 positions of guanines in opposite strands (3,7,9), and intrastrand crosslinks between two, presumably adjacent, guanines in the same strand (10-12).In bacteria (13-17), as well as in eukaryotic cells (18)(19)(20), a correlation between mutagenicity and antitumor activity ofseveral Pt compounds has been found, suggesting that lesions leading to mutation events can also be responsible for antitumor activity. Several...

In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation.

“…We found that all four dNTPs (lane 2) extend the substrate at only some sites and to lesser degree than in the presence of Mn (dimer position [35][36]. dATP is preferentially incorporated at some sites to a greater extent than with all four dNTPs present (dimer position [35][36]. Some insertion of dGTP occurs (dimer position 32-33); however, the extent is less and the number of sites is fewer than with dATP, as shown by the results in the presence of Mg2+ given in Table 1.…”

New clues for platinum antitumor chemistry: Kinetically controlled metal binding to DNA

“…The activity of cisplatin is closely related to its binding to DNA (35). Highly conclusive evidence for this target was the early observation that cells deficient in DNA repair are hypersensitive to cisplatin (36,37), suggesting that cisplatin and other drugs bind DNA. Early studies showed that of the four bases the N7 site of guanine is strongly preferred (see also Fig.…”

Alteration and activation of sequence-specific cleavage of DNA by bleomycin in the presence of the antitumor drug cis-diamminedichloroplatinum(II).

“…cis-DDP, on the other hand, binds covalently to DNA. Attachment of cis-DDP to poly(dG) regions through intrastrand crosslinking of guanosine N7 atoms is thought to be responsible for its various biological activities (13)(14)(15)(16)(17) DNA-Drug Reactions and Analysis. Reaction samples containing the 165-bp 32P-labeled DNA piece and sonicated calf thymus carrier DNA (1 mg/ml) were incubated with cis-or trans-DDP at a drug-to-nucleotide (D/N) mole ratio of 0.33, 0.165, 0.083, or 0.041 in 1 mM sodium phosphate/5 mM sodium chloride buffer, pH 7.4, at 37°C for 3 hr.…”

“…cis-DDP, on the other hand, binds covalently to DNA. Attachment of cis-DDP to poly(dG) regions through intrastrand crosslinking of guanosine N7 atoms is thought to be responsible for its various biological activities (13)(14)(15)(16)(17). Previously we showed that the intercalating drug ethidium bromide changes the nuclease-sensitive DNA-binding sites of cis-DDP (15) and that ethidium bromide, distamycin A, and actinomycin D modify the sequence-specific cleavage mode of bleomycin (18 (14).…”