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Topoisomerase II is required for the production of long Pol II gene transcripts in yeast.

ABSTRACT: The extent to which the DNA relaxation activities of eukaryotic topoisomerases (topo I and topo II) are redundant during gene transcription is unclear. Although both enzymes can often substitute for each other in vivo, studies in vitro had revealed that the DNA cross-inversion mechanism of topo II relaxes chromatin more efficiently than the DNA strand-rotation mechanism of topo I. Here, we show that the inactivation of topo II in budding yeast produces an abrupt decrease of virtually all polyA+ RNA transcripts of length above ? 3 kb, irrespective of their function. This reduction is not related to transcription initiation but to the stall of RNA polymerase II (Pol II) during elongation. This reduction does not occur in topo I mutants; and it is not avoided by overproducing yeast topo I or bacterial topo I, which relaxes (-) DNA supercoils. It is rescued by catalytically active topo II or a GyrBA enzyme, which relaxes (+) DNA supercoils. These findings demonstrate that DNA relaxation activities of topo I and topo II are not interchangeable in vivo. Apparently, only topo II relaxes efficiently the (+) DNA supercoils that stall the advancement of Pol II in long genes. A mechanistic model is proposed.

SUBMITTER: Joshi RS

PROVIDER: S-EPMC3439932 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Topoisomerase II is required for the production of long Pol II gene transcripts in yeast.

Joshi Ricky S RS Piña Benjamin B Roca Joaquim J

Nucleic acids research 20120619 16

The extent to which the DNA relaxation activities of eukaryotic topoisomerases (topo I and topo II) are redundant during gene transcription is unclear. Although both enzymes can often substitute for each other in vivo, studies in vitro had revealed that the DNA cross-inversion mechanism of topo II relaxes chromatin more efficiently than the DNA strand-rotation mechanism of topo I. Here, we show that the inactivation of topo II in budding yeast produces an abrupt decrease of virtually all polyA+ ...[more]

PMID: 22718977

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Project description:Dosage compensation refers to the equalization of most X-linked gene products between males and females. In Drosophila, it is mediated by the MSL complex that preferentially associates with numerous sites on the X chromosome in somatic cells of males, and is responsible for an enhancement of the transcriptional rate of a substantial number of X-linked genes. Here we show that topoisomerase II (Topo II) is an integral part of the mechanistic basis of dosage compensation and we highlight a novel function for this enzyme. A widely accepted model of transcription postulates that the moving bubble generated by an RNA polymerase elongating complex induces positive DNA supercoiling in the region ahead of the complex and negative supercoiling in its wake. These transitory changes in supercoiling are resolved by the action of topoisomerases. We have investigated the role of Topo II in dosage compensation by RNAi-mediated depletion, and we have used chromatin immunoprecipitation to determine its genomic distribution and relative abundance on X-linked genes. Topo II is enriched on dosage compensated genes and this enrichment is independent from the approximate two-fold enhancement in transcription of these genes. We have demonstrated an RNA-dependent association of Topo II with MLE, the ATPase-helicase subunit of the MSL complex. Our results indicate a role for Topo II that is additional to and different from its function in restoring normal DNA superhelicity during the transcription process. We suggest that the enhanced level of Topo II alters the DNA supercoiling of compensated gene units to facilitate transcription and could provide a basis for the recent report that the MSL complex enhances transcription by increasing the rate of elongation of RNA polymerase II. Investigation of dosage compensated gene expression levels in S2 and S2 Topo II RNAi knockdown cells. mRNA was isolated from S2 cells and S2 cells with RNAi knockdown of Topo II. The mRNA was then converted to cDNA and hybridized to a NimbleGen D. melanogaster gene expression array. 2 replicates each.

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Topoisomerase II is required for the production of long Pol II gene transcripts in yeast.

Publications

Topoisomerase II is required for the production of long Pol II gene transcripts in yeast.

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