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A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

ABSTRACT: We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis. Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily.

SUBMITTER: Castano IB

PROVIDER: S-EPMC145947 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

Castaño I B IB Heath-Pagliuso S S Sadoff B U BU Fitzhugh D J DJ Christman M F MF

Nucleic acids research 19960601 12

We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically d ...[more]

PMID: 8710513

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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.

Dataset Information

A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

Publications

A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

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