Role of DNA topoisomerase I in the transcription of supercoiled rRNA gene.

Abstract

The fraction DE-B obtained by fractionating an extract from rat mammary adenocarcinoma cells on a DEAE-Sephadex column was used for transcribing linear and supercoiled rRNA gene (rDNA). This fraction, which is known to contain RNA polymerase I and essential transcription factors, also contains DNA topoisomerase I activity. Inhibition of this topoisomerase activity by the selective inhibitor camptothecin markedly diminished transcription of supercoiled rDNA, and at a concentration of 150 ,uM, camptothecin almost completely inhibited DNA topoisomerase I activity and supercoiled rDNA transcription. Addition of exogenous calf thymus DNA topoisomerase I to the sample containing the drug restored the ability of the extract to transcribe supercoiled rDNA. Camptothecin, even at a concentration of 500 uM, had no significant effect on the transcription of linear rDNA. These studies show that relaxation of supercoiled rDNA by DNA topoisomerase I is essential for its transcription. The preferential inhibition of rRNA synthesis in vivo following treatment with camptothecin is probably due to selective camptothecin inhibition of DNA topoisomerase I activity.There has been considerable interest in understanding the role of supercoiled DNA in DNA replication and transcription (for a review, see ref. 1). Reports that supercoiling of cloned, circular prokaryotic chromosomes can activate the promoters of various genes in vitro (2, 3) have prompted studies to explore whether there is a similar mechanism in eukaryotic gene transcription. Several studies indicate that DNA supercoiling may have profound effects on gene expression in eukaryotes (3-7). Supercoiled DNA can be readily relaxed by DNA topoisomerases I and II, the enzymes that induce transient single-strand and double-strand breaks in DNA, respectively (1). DNA topoisomerase I is closely associated with the nucleolus (8, 9). However, it is not known whether relaxation of supercoiled rRNA gene (rDNA) by the type I DNA topoisomerase is obligatory to its transcription. We have been able to obtain a partially purified fraction from rat mammary adenocarcinoma cells that can accurately transcribe cloned rat rDNA (10). This fraction contains RNA polymerase I and a few accessory polypeptides, one of which has been subsequently identified as poly(ADP-ribose) polymerase (11). A similar fraction has also been obtained from nuclear extracts of normal rat liver and a rat hepatoma (12). We now report that this fraction contains DNA topoisomerase I that can control transcription of supercoiled rDNA. METHODSIn Vitro Transcription Assay. In vitro transcription was performed essentially as described (10). The whole cell extract derived from rat mammary adenocarcinoma cells was fractionated on a DEAE-Sephadex column, and the fraction designated DE-B that contains RNA polymerase I and essential transcription factors (10) was used in the assay. Supercoiled or Xho I-linearized plasmid (pB7-2.0) that consists of rat rDNA from position -167 to position +2000 (10) was used as template. The r...

Human tumor suppressor p14ARF negatively regulates rRNA transcription and inhibits UBF1 transcription factor phosphorylation

“…Moreover, p14 ARF and Topo I are both recovered in the chromatin fraction containing the rRNA promoter (Ayrault et al, 2004), in accordance with the requisite role of Topo I in rRNA transcription through the relaxation of supercoiled rDNA (Garg et al, 1987). We examined the possible involvement of p14 ARF in cell cycle arrest through both its activation of Topo I catalytic activity and inhibition of rRNA transcription.…”

“…We had previously described the existence of an interaction between p14 ARF and the topological enzyme Topoisomerase I (Topo I), essential in the transcription of supercoiled rRNA genes (Garg et al, 1987;Karayan et al, 2001;Ayrault et al, 2003). More recently, we have demonstrated by ChIP experiments that p14 ARF protein was able to specifically interact with the human rRNA promoter in the presence of Topo I (Ayrault et al, 2004).…”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…A number of studies have indicated that this enzyme which interconverts topological isomers of DNA by breaking and resealing one DNA strand, is required for proper transcription ofrRNA genes (e.g. Garg et al, 1987;Egyhazi and Durban, 1987;Rose et al, 1988;Zhang et al, 1988;Kim and Wang, 1989). Topoisomerase I has been shown to be associated with RNA polymerase I and is located within the transcribed region of the rRNA genes but absent from the spacer region (Zhang et al, 1988).…”

Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription

“…Although their biological function is not completely defined , this class of topoisomerase has been implicated in transcriptional (Akrigg and Cook 1980;Dynan and Burgess 1981;Sternglanz et al 1981;Weisbrod 1982;Fleischmann et al 1984 ;Gilmour et al 1986;Shastry 1986) as well as in recombinational and replicational (Kikuchi and Nash 1979 ;Sternglanz et al 1981 ; also see Gellert 1981 ;Bullock et al 1985 ;Zeng et al 1985) events. J n higher eukaryotes topoisomerase I is enriched in the nucleolus (Fleischmann et al 1984;Muller et al 1985) and may play a role in transcription of supercoiled rDNA in vitro (Garg et al 1987). Studies in Tetrahymena indicate that the ribosomal DNA-associated enzyme…”