Comparison of benzo[a]pyrene-diol-epoxide binding to histone H2A with different carboxy-terminal regions.

We have compared (+/-)-7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) binding to three vertebrate histones H2A with different C-terminal regions. HPLC analyses of core histones prepared from nuclei exposed to [3H]BPDE-I showed that rat liver and chicken erythrocyte histones H2A were heavily labeled by [3H]BPDE-I, but Xenopus laevis liver histone H2A was not. This result was confirmed by HPLC analyses of V8-protease digests of BPDE-I bound to histone H2A purified from the three different nuclei. There are significant amino acid sequence differences only in the C-terminal regions of the different histones H2A, where rat liver and chicken erythrocyte histones H2A contain two and one histidine residues, respectively, while the amino acid sequence of Xenopus histone H2A contains no histidine. Pre-treatment of the in situ BPDE-I-modified H2A.2 from rat liver with carboxypeptidase B, which should remove the C-terminal lysine from the protein, resulted in increased retention times on reverse-phase HPLC for the adduct-containing peptides upon subsequent V8-protease digestion. This suggested that the site(s) of BPDE-I modification are located primarily in the C-terminal octapeptide of rat H2A.2. To confirm this, C-terminal V8-peptides of the different histones H2A were isolated and reacted with BPDE-I at physiological pH in vitro. The HPLC analyses of the reaction mixtures indicated that the C-terminal peptide of rat liver and chicken erythrocyte histones H2A was a target site for BPDE-I binding in nuclei. It is suggested that the nucleophilic target amino acid for BPDE-I binding in histone H2A may be a histidine located close to the C terminus.