Linear DNA does not form chromatin containing regularly spaced nucleosomes.

Abstract

The topological state of DNA may play a role in regulating chromatin structure and gene expression in eucaryotes. To test this hypothesis, the arrangements of nucleosomes on circular and unit-length linear simian virus 40 (SV40) DNAs incubated in nuclei of Xenopus oocytes were determined by (i) analyzing changes in the electrophoretic properties of the DNAs and (ii) examining the patterns of DNA fragments resulting from digestions with micrococcal nuclease. Whereas circular DNA became associated with nucleosomes that were arranged along the DNA at regular intervals of approximately 195 base pairs, linear DNA failed to reconstitute into chromatin containing regularly spaced nucleosomes. DNA that failed to form proper chromatin was gradually degraded, indicating that histone proteins in proper association with DNA may be the cellular component that normally protects chromosomal DNA from endonucleolytic attack. When either circular or linear DNA was incubated in an in vitro transcription system made from a whole-cell extract of HeLa cells, most of the molecules did not associate with histone proteins to form regularly spaced nucleosomes. Furthermore, linearization of mRNA-encoding DNAs, including SV40, reduces their transcriptional activity in Xenopus oocytes to a level comparable to that obtained with the in vitro transcription system employed here. Therefore, proper association of DNA with appropriate cellular chromosomal factors may be a prerequisite for proper transcription by RNA polymerase II.