Studies on the ATP requirements of in vitro chromatin assembly

To gain a more complete understanding of the process of in vitro chromatin assembly, an examination of the energy requirements of nucleosome formation must be undertaken. The experiments outlined in this manuscript address this issue by making use of the Xenopus laevis S-150 cell-free extract. The S-150 catalyzes chromatin assembly on circular DNA templates dependent either on the exogenous addition of ATP or regeneration of endogenous ATP. We define two distinct, but temporally ordered, phases of the overall process. The first, nucleosome formation, occurs in the presence of endogenous levels of ATP, while the second phase, chromatin assembly, which we define as the development of properly spaced nucleosomes, requires a higher level of ATP. Both phases lead to a distribution of molecules with similar superhelical densities. Taken together, these data suggest that chromatin assembly may consist of two distinct steps differing in their strategy cofactor requirement. The experiments presented in this manuscript support the concept that nucleosomes first assemble, perhaps randomly, on the DNA and are gradually matured into a canonical chromatin structure with periodic spacing.