Assembly of SV40 chromatin in a cell-free system from Xenopus eggs

Abstract

A cell-free system is described which assembles chromatin from purified DNA in 1 hr under physiological incubation conditions. It consists of a 145,000 x g (maximum) supernatant fraction from eggs of Xenopus laevis. It converts SV40 DNA to a nucleoprotein which co-sediments with naturally occurring SV40 chromatin and which can be cleaved by micrococcal nuclease to a highly ordered pattern of DNA fragments resembling those from digestion of liver chromatin. It inserts superhelical turns into relaxed, covalently closed DNA. The assembly process is not cooperative. Under limiting conditions, each DNA molecule becomes partially assembled. Assembly does not require replication of the DNA or protein synthesis, but occurs from a stored histone pool of at least 40 ng per egg. Under conditions of DNA excess, assembly becomes dependent upon the amount of exogenous histones added to the incubation. Apart from histones and a nicking-closing activity, chromatin assembly requires an additonal thermolabile factor which is present in the egg supernatant.