NAP1 Catalyzes the Formation of either Positive or Negative Supercoils on DNA on Basis of the Dimer−Tetramer Equilibrium of Histones H3/H4

Abstract

We have studied the tetramer-dimer equilibrium of histones H3/H4 and its effect on DNA supercoiling. Two approaches were found to shift the equilibrium toward dimer. In both instances, when deposited on DNA, the dimers formed positively coiled DNA. The first approach was to modify cysteine 110 of H3 with 5,5'-dithio-bis(2-nitrobenzoic acid (DTNB) and to directly add the histones to DNA at physiological ionic strength. The second approach involved adding an excess of the histone chaperone, nucleosome assembly protein 1 (NAP1) to the H3/H4 prior to deposition on the DNA. It was also observed that when H3/H4 were deposited in the tetrameric state, negatively coiled DNA was formed. The topological state of the DNA prior to deposition was also found to influence the final conformational state of H3/H4. It is proposed that in the tetrameric state, the H3-H3 interface has a left-handed pitch prior to binding DNA. In the dimeric state, the H3-H3 interface is not established until bound to DNA, at which point either the left or right-handed pitch will form on the basis of the initial topology of the DNA. Formaldehyde cross-linking and reversal were applied to identify the histone-histone interactions that facilitate the formation of positive stress. Higher-order interactions between multiple H3/H4 dimers were required to propagate this specific conformation. Changes in the conformational state of H3/H4 were also observed when the histones were bound to DNA prior to treatment with NAP1. It is proposed that these conformational changes in H3/H4 are involved in promoter activation and transcription elongation through nucleosomes.