Probing interactions between lysine residues in histone tails and nucleosomal DNA via product and kinetic analysis.

Abstract

The histone proteins in nucleosome core particles are known to catalyze DNA cleavage at abasic and oxidized abasic sites, which are produced by antitumor antibiotics and as a consequence of other modalities of DNA damage. The lysine rich histone tails whose post-translational modifications regulate genetic expression in cells are mainly responsible for this chemistry. Cleavage at a C4′-oxidized abasic site (C4-AP) concomitantly results in modification of lysine residues in histone tails. Using LC-MS/MS, we demonstrate here that that Lys8, -12, -16, and -20 of histone H4 were modified when C4-AP was incorporated at a hot spot (superhelical location 1.5) for DNA damage within a nucleosome core particle. A new DNA–protein cross-linking method that provides a more quantitative analysis of individual amino acid reactivity is also described. DNA–protein cross-links were produced by an irreversible reaction between a nucleic acid electrophile that was produced following oxidatively induced rearrangement of a phenyl selenide derivative of thymidine (3) and nucleophilic residues within proteins. In addition to providing high yields of DNA–protein cross-links, kinetic analysis of the cross-linking reaction yielded rate constants that enabled ranking the contributions by individual or groups of amino acids. Cross-linking from 3 at superhelical location 1.5 revealed the following order of reactivity for the nucleophilic amino acids in the histone H4 tail: His18 > Lys16 > Lys20 ≈ Lys8, Lys12 > Lys5. Cross-linking via 3 will be generally useful for investigating DNA–protein interactions.