Frequencies of mutagenic translesion DNA synthesis over cisplatin-guanine intra-strand crosslinks in lacZ plasmids propagated in human cells

• Plasmids site-specifically carrying cisplatin-guanine crosslinks were constructed.
• Frequency of mutations through TLS over the crosslinks was measured in human cells.
• Pt-GTG crosslink is more mutagenic than Pt-GG crosslink in human cells.
• Most mutations were induced at 5′ G residue in the Pt-GTG crosslink.
• Pt-GTG crosslink becomes an effective obstacle for cancer cell division.

Cisplatin (cis-diamminedichloroplatinum(II)), a widely used anticancer drug, forms inter- and intra-strand DNA crosslinks. The major intra-strand crosslinks are Pt adducts at 1,2-d(GpG) and 1,3-d(GpNpG) (Pt-GG and Pt-GNG, respectively). Although most of the intra-strand crosslinks are removed by the nucleotide excision repair (NER), the remaining crosslinks can cause mutations through the translesion DNA synthesis (TLS) during chromosome replication. To understand the precise mechanism of cisplatin mutagenesis in human cells, the plasmid carrying a single Pt-GG or 1,3-d(GpTpG) crosslink (Pt-GTG) site-specifically in lacZ gene was constructed and propagated in NER-defective xeroderma pigmentosum cells. The plasmids retrieved from the cells were introduced into indicator bacterial cells to access frequencies of TLS and mutations. The experiments revealed that Pt-GTG blocked DNA replication more strongly and caused more mutations (29.1%) than Pt-GG (1.7%). Most mutations were G to A or T base changes at 5′ G residue in the Pt-GTG crosslinks. These results indicate that the Pt-GTG crosslinks become effective obstacles for cancer cell division, and have an important role for cisplatin cancer therapy.