A 1,2-d(GpG) cisplatin intrastrand cross-link influences the rotational and translational setting of DNA in nucleosomes.

The mechanism of action of platinum-based anticancer drugs such as cis-diamminedichloroplatinum(II), or cisplatin, involves three early steps: cell entry, drug activation, and target binding. A major target in the cell, responsible for the anticancer activity, is nuclear DNA, which is packaged in nucleosomes that comprise chromatin. It is important to understand the nature of platinum-DNA interactions at the level of the nucleosome. The cis-{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand cross-link is the DNA lesion most commonly encountered following cisplatin treatment. We therefore assembled two site-specifically platinated nucleosomes using synthetic DNA containing defined cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-links and core histones from HeLa-S3 cancer cells. The structures of these complexes were investigated by hydroxyl radical footprinting and exonuclease III mapping. Our experiments demonstrate that the 1,2-d(GpG) cross-link alters the rotational setting of the DNA on the histone octamer core such that the lesion faces inward, with disposition angles of the major groove relative to the core of xi approximately -20 degrees and xi approximately 40 degrees . We observe increased solvent accessibility of the platinated DNA strand, which may be caused by a structural perturbation in proximity of the 1,2-d(GpG) cisplatin lesion. The effect of the 1,2-d(GpG) cisplatin adduct on the translational setting of the nucleosomal DNA depends strongly on the position of the adduct within the sequence. If the cross-link is located at a site that is in phase with the preferred rotational setting of the unplatinated nucleosomal DNA, the effect on the translational position is negligible. Minor exonuclease III digestion products in this substrate indicate that the cisplatin adduct permits only those translational settings that differ from one another by integral numbers of DNA helical turns. If the lesion is located out of phase with the preferred rotational setting, the translational position of the main conformation was shifted by 5 bp. Additionally, a fraction of platinated nucleosomes with widely distributed translational positions was observed, suggesting increased nucleosome sliding relative to platinated nucleosomes containing the 1,3-intrastrand d(GpTpG) cross-link investigated previously (Ober, M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 6278-6286).

Cisplatin damage overrides the predefined rotational setting of positioned nucleosomes.

Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24 degree. The intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.

Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase.

Aerobic respiration generates reactive oxygen species that can damage guanine residues and lead to the production of 8-oxoguanine (8oxoG), the major mutagenic oxidative lesion in the genome. Oxidative damage is implicated in ageing and cancer, and its prevalence presents a constant challenge to DNA polymerases that ensure accurate transmission of genomic information. When these polymerases encounter 8oxoG, they frequently catalyse misincorporation of adenine in preference to accurate incorporation of cytosine. This results in the propagation of G to T transversions, which are commonly observed somatic mutations associated with human cancers. Here, we present sequential snapshots of a high-fidelity DNA polymerase during both accurate and mutagenic replication of 8oxoG. Comparison of these crystal structures reveals that 8oxoG induces an inversion of the mismatch recognition mechanisms that normally proofread DNA, such that the 8oxoG.adenine mismatch mimics a cognate base pair whereas the 8oxoG.cytosine base pair behaves as a mismatch. These studies reveal a fundamental mechanism of error-prone replication and show how 8oxoG, and DNA lesions in general, can form mismatches that evade polymerase error-detection mechanisms, potentially leading to the stable incorporation of lethal mutations.