TFIIIA and DNA supercoiling: toward resolving a controversy.
DNA supercoiling in eukaryotes is mediated by the process of chromatin assembly. This process couples the binding and wrapping of DNA around nucleosomal core particles and the enzymatic activity of DNA topoisomerase(s). Kmiec and Worcel (1985) reported that the binding of an RNA polymerase III transcription factor (TFIIIA) could accelerate DNA supercoiling catalysed by a Xenopus laevis cell-free extract. Although the reaction was repeatedly reproduced in the parent laboratory by numerous workers, another laboratory, interested in the molecular biology of TFIIIA, failed to reproduce TFIIIA-mediated supercoiling or gyration. In this review, an attempt is made to transcend personal beliefs and describe the experimental approaches used to clarify this issue. The original variability lay in the amount of endogenous RNA within the cell free extract and the concentrations of MgCl2 used to conduct the experiments.
Kmiec
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EB
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002474:Cell-Free System; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D015636:Magnesium Chloride / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009865:Oocytes; D012313:RNA / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*; D014982:Xenopus laevis
D004278:DNA, Superhelical; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D015636:Magnesium Chloride; D012313:RNA; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I