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.Eukaryotic cells have two major topoisomerases that are capable of efficiently relaxing torsionally stressed DNA: topoisomerase I (Top1) and topoisomerase II (Top2) (75). They are both abundant nuclear proteins with roles in many DNA activities, and since they both can relax positive and negative torsion, they can substitute for each other in most situations (11,28,29,35,62). In spite of this partial functional redundancy, they control DNA topology by very different mechanisms (65). Top1 (a type IB topoisomerase) makes transient single-strand breaks in torsionally stressed DNA (recognizing the torque in such DNA), followed by controlled rotation of the nicked strand and resealing of the DNA in a more relaxed state (38). Top2 (a type IIA topoisomerase) recognizes juxtaposed DNA helices (as in supercoiled DNA) and passes one DNA helix through the other by making a transient doublestrand break in one of the helices (61, 65

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…Positive supercoils generated by heavy Pol I transcription might accumulate in NTS1 where they could facilitate silencing by RENT at the RFB. Indeed, topoisomerase I is required for rDNA silencing, and a lack of TOP1 results in accumulation of negative supercoiling in the rDNA genes, presumably due to the selective relaxation of positive supercoils by another topoisomerase French et al 2011). Also consistent with a supercoiling model, transcriptional elongation by Pol I was shown to be necessary for downstream silencing (Buck et al 2016).…”

Topoisomerase 1 and Single-Strand Break Repair Modulate Transcription-Induced CAG Repeat Contraction in Human Cells

“…In bacteria the link has been ascribed to the effects of negative supercoiling on enhancing the formation of repeat-induced non-B DNA structures (38), which are thought to constitute the key common event leading to changes in repeat-tract length (24,40,56). The negative supercoiling that develops behind a transcribing RNA polymerase (54) would provide a natural connection between transcription and repeat instability and could explain our results with TOP1 inhibitors, especially with siRNA knockdowns of TOP1, which might be expected to increase supercoiling stress (9).…”

Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

“…In addition, replication bubbles with a length of at least 10 kb were reported to occur in yeast (25). Therefore, similar replication bubbles should also be generated in A. oryzae.…”