Nucleic Acids Research

Nucleosome-cores were reconstituted by the salt-dialysis method onto closed circular pDHgl6 DNA which contains a d(CG/GC)12 sequence. Alternating d(CG/GC)n sequences form left-handed Z-DNA readily when contained in negatively supercoiled DNA. We have investigated the ability of the d(CG/GC)12 sequence to be organized into nucleosome-cores when stabilized as Z-DNA through negative supercoiling. We have found that nucleosome assembly at the d(CG/GC)12 insert is prevented when the sequence is stable in the Z-conformation but it is not affected at all when the sequence adopts the righthanded B-form. INTRODUCTION DNA in solution may exist under a variety of dif ferent structural conformations (1). DNA sequences containing alternating purine and pyrimidine residues are known to undergo transition to the left-handed Z-DNA conformation in response to changing environmental conditions (2). In particular, the repetitive d(CG/GC)n sequence has been shown to form Z-DNA when contained in negatively supercoiled DNA (3,4). Other alternating (5,6) and non-strictly alternating (7) DNA sequences can also form Z-DNA through negative supercoiling. Although no direct evidence for its in vivo existence has yet been obtained, it has been suggested that the left-handed Z-conformation may play an important structural or regulatory function in eukaryotic cells (8). Recent results strongly suggest that Z-DNA is formed transiently during homologous DNA recombination (9). On the other hand, a class of nuclear proteins exists which preferentially recognize the left-handed Z-conformation (10-12). In particular, a protein factor which mediates homologous DNA recombination in Ustilago has been shown to be a Z-DNA ©) I R L Press Limited, Oxford, England. 8899 Nucleic Acids Research binding protein (13). Eukaryotic DNA is organized as chromatin. DNA in chromatin is tightly packed into nucleosome-cores which constitute the basic repetitive structural subunit of chromatin organization. Considerations of the possible biological role of Z-DNA in eukaryotes raise the question of the effect of this alternative DNA conformation on the nucleosomal organization of chromatin. In this paper, nucleosome-cores were reconstituted in vitro by the salt dialysis method onto closed circular pDHgl6 DNA which contains an alternating d(CG/GC)12 sequence stabilized in the left-handed Z-conformation through negative supercoiling. The ability of the alternating d(CG/GC)12 sequence to be organized into nucleosomes has been investigated. Others (14,15) have also addressed this question by studying nucleosome reconstitution onto the synthetic chemically modified poly(dGm5dC) copolymer stabilized in the left-handed Z-conformation by divalent or multivalent cations. We have found that nucleosome reconstitution at the d(CG/GC)12 sequence is prevented when the sequence forms Z-DNA but it is not affected at all when the alternating d(CG/GC)12 sequence is in the right-handed Bform. The biological significance of these results is discussed. Short stretches of alternating d(CG/GC)n sequences are moderately abundant in eukaryotic genomic DNA (16) and they have been shown to enhance homologous recombination of adjacent sequences by as much as 15 fold (17). On the other hand, negative supercolling is likely to be an important factor determining Z-DNA stability in vivo. HATERIALS AND METHODS Histones and DNA Core-histones were obtained from purified chicken erythrocyte nuclei by differential acid extraction according to Johns (18). Purified core-histones were kept at -200C as an acetone powder. HI/H5 or HMGs proteins were absent and the four corehistones were present to approximately equimolecular amounts. pDHgi6 is a 2.2 kb plasmid derived from pBR322 which contains a d(CG/GC)12 insertion (19). Plasmid DNA was prepared by standard procedures (20). The linking difference (T) of