title: Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes.
abstract: To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.
authors.affiliation: Department of Microbiology, Box 800734, University of Virginia Health System, Charlottesville, VA 22908-0734, USA. alb4h@virginia.edu
authors.forename: Sarah L
authors.initials: SL
authors.lastname: French
authors.forename: Martha L
authors.initials: ML
authors.lastname: Sikes
authors.forename: Robert D
authors.initials: RD
authors.lastname: Hontz
authors.forename: Yvonne N
authors.initials: YN
authors.lastname: Osheim
authors.forename: Tashima E
authors.initials: TE
authors.lastname: Lambert
authors.forename: Aziz
authors.initials: A
authors.lastname: El Hage
authors.forename: Mitchell M
authors.initials: MM
authors.lastname: Smith
authors.forename: David
authors.initials: D
authors.lastname: Tollervey
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Smith
authors.forename: Ann L
authors.initials: AL
authors.lastname: Beyer
mesh_terms: D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004789:Enzyme Activation; D005800:Genes, Fungal / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D013314:Stress, Mechanical; D054159:Torsion, Mechanical; D014158:Transcription, Genetic
chemical_list: D004256:DNA Polymerase I; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II