Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast.

Abstract

In Saccharomyces cerevisiae cells harboring a GAL) promoter-linked ,-galactosidase gene, the simultaneous expression of Escherichia coi DNA topolsomerase I and inactivation of yeast DNA topoisomerases I and II reduces the cellular level of P-galactosidase to an undetectable levd. Analysis of intracellular mRNA level and the density of RNA polymerase along DNA indicates that this reduction is due to the suppression of transcription and that both plasmid-borne and chromosomally located genes are affected. These results are interpreted in terms of inhibition of transcription in vivo due to positive supercoiling of the DNA template: preferential removal of transcription-generated negative supercopls by E. colU DNA topoisomerase I in the absence of both yeast DNA topoisomerases I and II results in the accumulation of positive supercoils in intracellular DNA. In normal prokaryotic or eukaryotic cells, accumulation of positive supercolls is presumably avoided through the balanced actions of DNA topoisomerases.In prokaryotes, there is substantial evidence that gene expression is often influenced by the cellular levels of DNA topoisomerases and, by implication, the state of supercoiling of intracellular DNA (1-3). Whether the same is true in eukaryotes is less clear. There is evidence that in eukaryotic as well as prokaryotic cells, transcription may lead to supercoiling ofthe template DNA, and one ofthe functions ofDNA topoisomerases is the removal of supercoils (4,5). Eukaryotic DNA topoisomerase I is known to be preferentially associated with actively transcribed regions of chromatin, and this association is generally assumed to reflect a functional role of the enzyme in transcription (2,(6)(7)(8). Yeast mutants devoid of the enzyme exhibit no abnormality in transcription, however (9). In these top] mutants, the normal roles of DNA topoisomerase I might be fulfilled by DNA topoisomerase II, as both enzymes are capable of relaxing positively and negatively supercoiled DNA in vivo (5, 10). Simultaneous inactivation of DNA topoisomerases I and II, through the use of topi top2 double-mutants, reduces rRNA synthesis by a factor of 10 and total poly(A)+ RNA synthesis by a factor of 3 (11, 12). These reductions can be attributed to changes in the topology of the DNA template, but a direct link is difficult to establish because of the multiple cellular roles of the topoisomerases. A causal relation between template topology and proficiency is further clouded by the finding that transcription of several specific Saccharomyces cerevisiae genes appears to be unaffected by inactivation of both DNA topoisomerases I and 11 (13 11461The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.

“…It has been shown that both polymerase I (48) and polymerase II (46) transcription in yeast cells can stimulate recombination. In addition, increased positive supercoiling, resulting from the overexpression of the bacterial TopA protein in yeast cells, diminishes transcription (16). Therefore, if a reverse gyrase were used to regulate transcription by introducing positive supercoils, then the absence of such an activity would lead to an increase in transcription and thus stimulate recombination.…”

Hpr1 Is Preferentially Required for Transcription of Either Long or G+C-Rich DNA Sequences in Saccharomyces cerevisiae

“…The cleavage of R-loops by specific nucleases might initiate class-switch recombination (58), providing a mechanism to explain transcription-associated recombination. Nothing is known about the influence of RNA-DNA structures on RNAPII-dependent transcription (20). However, it has been proposed that R-loops formed during rRNA transcription elongation in E. coli constitute roadblocks for the next transcribing RNA polymerase (25).…”

Chromatin dynamics during DNA replication

“…These stresses are relieved by DNA topoisomerases (Wang 2002). Notably, early studies have shown that topoisomerases relieve transcription inhibition caused by positive supercoiling, and implicated histone acetylation in this process (Krajewski and Luchnik 1991;Gartenberg and Wang 1992). We therefore profiled H3K9ac during replication also in strains depleted of topoisomerases (Wang 2002).…”