The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation.

Puja Yadav,Nayun Kim,Norah Owiti

doi:10.1093/nar/gkv1152

Puja Yadav, Nayun Kim + Show 1 more

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https://doi.org/10.1093/nar/gkv1152

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Abstract

Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation.

Highlights

Genome instability––recombination and mutagenesis––is elevated by highly active transcription [1,2,3,4,5,6,7]
We present a clear in vivo demonstration of sequencespecificity in R-loop accumulation; when the G4-forming S␮ is highly transcribed, RNA:DNA hybrids accumulate in an orientation-specific manner that correlates with the orientation-specific increase in genome instability observed in Top1- or RNase H-deficient genetic backgrounds
We previously reported on the construction of recombination reporter assay that allowed us to determine the contribution of guanine-run containing sequences to genome instability under high- or low-transcription conditions [44]

Summary

Introduction

Genome instability––recombination and mutagenesis––is elevated by highly active transcription [1,2,3,4,5,6,7] (see [8,9] for review). The unwinding of the duplex DNA, mandatory for transcription process, transiently produces single stranded DNA regions of the non-transcribed strand (NTS) that are highly susceptible to chemical and enzymatic modifications. This effect can be aggravated when the nascent RNA stably anneals to the template DNA strand (transcribed strand – TS) producing extensive stretches of RNA:DNA hybrids or R-loops [13]. R-loops lead to genome instability by obstructing DNA replication [14] or by generating DSB via structure specific endonucleotic activity [15]. Extensive R-loop accumulation, at the highly transcribed rDNA locus and the tRNA genes, is observed in absence of this family of enzymes [22,23]

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