Thermodynamic and mechanistic insights into translesion DNA synthesis catalyzed by Y-family DNA polymerase across a bulky double-base lesion of an antitumor platinum drug

To determine how the Yfamily translesion DNA polymerase h (Polh) processes lesions remains fundamental to understanding the molecular origins of the mutagenic translesion bypass. We utilized model systems employing a DNA double-base lesion derived from 1,2-GG intrastrand crosslinks of a new antitumor PtII complex containing a bulky carrier ligand, namely [PtCl2(cis-1,4-dach)] (DACH= diaminocyclohexane). The catalytic efficiency of Polh for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the 1,2-GG cross-link was markedly reduced by the DACH carrier ligand. This reduced efficiency of Polh to incorporate the correct dCTP could be due to a more extensive DNA unstacking and deformation of the minor groove induced in the DNA by the cross-link of bulky [PtCl2(cis-1,4-dach)]. The major products of the bypass of this doublebase lesion produced by [PtCl2(cis-1,4-dach)] by Polh resulted from misincorporation of dATP opposite the platinated G residues. The results of the present work support the thesis that this misincorporation could be due to sterical effects of the bulkier 1,4-DACH ligand hindering the formation of the Polh–DNA–incoming nucleotide complex. Calorimetric analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dATP by Polh as well.