In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.
Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.
Department of Molecular Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University School of Medicine, Philadelphia, PA 19107.
J M
JM
Sekiguchi
E B
EB
Kmiec
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D015636:Magnesium Chloride / Q000494:pharmacology; D009707:Nucleosomes / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014982:Xenopus laevis
D002457:Cell Extracts; D002843:Chromatin; D004270:DNA, Circular; D004278:DNA, Superhelical; D009707:Nucleosomes; D015636:Magnesium Chloride; D012313:RNA; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D012259:Ribonuclease, Pancreatic