Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile

Abstract

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.

Figure 1.

BisQM alkylation of DNA in the absence and presence of the histone octamer and after assembled into a NCP. Samples were treated with increasing concentrations of bisQM (0, 5, 50, 100, 250, 500 μM) at 4 °C for 24 h and then treated with piperidine to identify sites of alkylation. The resulting fragments were separated by denaturing gel electrophoresis (10% PAGE) and visualized by phosphorimagery.

Figure 2.

Formation of DNA-histone cross-links by treating the NCP with bisQMP. Individual samples of the NCP were treated with the indicated concentrations of bisQMP at 4 °C and quenched after 24 h by freezing in liquid N₂ as described in the Materials and Methods section. Samples were then thawed for 5 min at room temperature and divided into two sets. One set was analyzed directly, and the second set was treated with proteinase K (2 μL, 1.6 U) for 15 min at room temperature. All samples were then combined with loading dye, separated by 10% denaturing SDS-PAGE, and visualized by phosphorimagery.

Figure 3.

Peptide-QM adducts formed by the histone octamer. UPLC–MS analysis of the tryptic peptides formed by the histone octamer after treatment with (A) monoQMP and (B) bisQMP. Total ion count was monitored during elution of the peptides before (black) and after (red) reaction with the QMs. Signals unique to the alkylated samples are labeled according to Table 1. Signals labeled with “0” do not contain [M + H]⁺ values that correspond to a QM adduct or peptide fragment. Peptide coverage by this analysis is summarized in Figure S3. (C) MS² spectrum of the adduct formed between monoQM and TESSK of histone H2A (Table 1, entry I). (D) MS² spectrum of the adduct formed between bisQM and SAK of histone H2A (Table 1, entry IV).

Figure 4.

Reconstitution of NCP after alternative treatment of (A) 601 DNA and (B) histone octamer with bisQM. Lanes labeled as DNA contain DNA prior to reconstitution and lanes labeled NCP contain the products generated after reconstitution with the histone octamer. The relative distribution of species separated by native PAGE (6%) was measured by phosphorimaging the [³²P]-labeled DNA.

Figure 5.

Location of protein adducts formed in NCP (PDB: 1KX5) from alkylation by monoQM and bisQM.

Scheme 1.

(A) General Strategy for Fluoride-Induced Generation of QM and Its Subsequent Alkylation of Nucleophile (Nuc); (B) tert-Butyl dimethylsilyl-Protected (TBDMS) Quinone Methide Precursors (MonoQMP and BisQMP) Used To Generate Electrophilic MonoQM and BisQM, Respectively

Scheme 2.

Detecting QM Transfer from DNA to Protein