Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles

Abstract

C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

Figure 1

General structure features of a nucleosome core particle. (A) Structural representation of one-half of the NCP (PDB: 1kx5). Modified nucleotides are shown as red spheres. (B) Amino acid sequence of histone H4 tail. (C) Amino acid sequence of histone H3 tail.

Figure 2

Generic structure of nucleosomes containing DOB in the linker DNA region.

Figure 3

Reactivity of DNA abasic sites at position 89 in NCPs within 601 DNA compared to in free DNA of the same sequence.

Figure 4

Time-dependent DPC formation from DOB at three positions within NCPs in the absence (A) or presence of (B) NaBH₃CN.

Figure 5

MALDI-TOF mass spectra of peptide fragments obtained from in-gel acetylation and trypsin digest of (A) WT histone H4 and (B) the DPC produced by DOB₈₉. (The range of amino acids corresponding to each peptide is in parentheses.) (C) Peptide sequences and respective calculated m/z.

Figure 6

Proximity of histone H4 tail to nucleotide 89 in the vicinity of SHL 1.5. Proximal lysine residues are shown as spheres (PDB: 1kx5).

Figure 7

Effects of mutated histone H4 on the reactivity of DOB₈₉ in NCPs.

Figure 8

MALDI-TOF mass spectrum of peptide 13–20 (calcd m/z = 975.1220) of modified histone H4 protein digested by Lys C. K* = 6.

Figure 9

Peptide mapping of (A) fragment 9–16 obtained from Lys C digest and (B) fragment 20–23 from trypsin digest of H4 from NCP containing DOB₈₉. K* = 6.

Figure 10

Effects of tailless histone proteins on the reactivity of DOB₁₅₉ and DOB₁₇₆.

Figure 11

Modification of Lys4 by DOB₁₅₉. (A) MALDI-TOF mass spectrum of modified histone H3 digested by Lys C (calcd m/z = 1125.297). (B) LC-MS/MS analysis on fragment 3–8 obtained from trypsin digest of modified histone H3. K* = 6.

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