The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1-mediated nucleosome assembly onto replicated DNA in vitro

An in vitro reconstitution system for the analysis of replication-coupled n ucleosome assembly is described. In this "two-step system," nucleosome asse mbly is performed in a separate reaction from DNA replication, wherein puri fied newly replicated DNA remains noncovalently marked for subsequent chrom atin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the n ucleosome assembly is performed separately from the DNA replication step, t his system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-t erminal domains of histones H3 and H4 play an important but redundant funct ion in nucleosome assembly in the budding yeast, Saccharomyces cerevisiae. It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-l- mediated formation of nucleosome cores onto newly replicated DNA in vitro. CAF-1 and each of its individual subunits stably bound to recombinant (H3.H 4)(2) tetramers lacking the N-terminal domains of both H3 and H4. Therefore , the N-terminal tails of histone H3 and H4 that contain the specific acety lation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required fo r a CAF-1 independent pathway or function after deposition, by marking of n ewly replicated chromatin.