Competition for formation of nucleosomes on fragmented SV40 DNA: a hyperstable nucleosome forms on the termination region

Abstract

We have studied the relative abilities of different simian virus 40 (SV40) DNA segments to reconstitute into nucleosomes in vitro. The SV40 genome was separated into 15 discrete fragments by restriction endonuclease digestion and reconstituted with calf thymus core histones under conditions of varying histone-to-DNA ratios. Three fragments show very different abilities to form nucleosomes when low histone-to-DNA ratios require all fragments to compete for available histones. Two of these fragments, both from within protein-coding regions, are significantly underreconstituted. The third fragment, covering the SV40 termination region, competes much more effectively for histones than the other 14 fragments. The fragment containing the SV40 origin region formed nucleosomes with about average probability. Overall, the SV40 fragments differed by approximately an order of magnitude in their abilities to support nucleosome formation in vitro. The stability of the nucleosomes was measured by challenge with high concentrations of the destabilizing reagent heparin. The fragment that reconstituted most effectively also formed nucleosomes that were unusually stable to heparin challenge. These observations are intriguing since this fragment contains the sequences where replication of SV40 DNA commonly terminates and where early messenger RNA synthesis may terminate as well. The existence of unique hyperstable nucleosomes in this region suggests the interesting possibility that such nucleosomes may assist in termination events by assisting in the pausing of replication or transcription complexes.