Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae.

Éva Hegedüs,Aziz El Hage,László Halász,Yves Pommier,Viktor Dombrádi,Martin R Webb,Endre Kókai,Gábor Szabó,Rozenn Jossé,Zsuzsa Antunovics,Lóránt Székvölgyi,László Imre,Péter Nánási

doi:10.1093/nar/gky743

Abstract

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3′OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3′OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

Highlights

Recent observations in several mammalian experimental models suggest that transcriptional activation of RNA polymerase II (RNAP II) dependent genes frequently involves formation of DNA strand breaks, supposedly elicited by topoisomerase 2␤ (Top 2␤) [1,2,3,4,5,6,7]
The nicks are distributed at ≤ 200 kb distance apart from each other on each strand, so as to yield ∼70–100 kb ds fragments upon further breakage at these predilection points. These findings extend our earlier reports on loop-size DNA fragmentation [25,36,37,85] as: (i) The nick-character of the breaks was corroborated by their independent distribution on the two DNA strands and in molecular combing experiments. (ii) By excluding superhelical tension as a factor eliciting the breaks, what further confirmed their endogenous nature. (iii) By determining the distribution of the nicks along both the whole genomic DNA (gDNA) and the rDNA in WT and S. cerevisiae mutants
The loop-size periodicity was recapitulated in the rDNA cluster, implicating sequence-unrelated factors in their generation/maintenance

Summary

Introduction

Recent observations in several mammalian experimental models suggest that transcriptional activation of RNA polymerase II (RNAP II) dependent genes frequently involves formation of DNA strand breaks, supposedly elicited by topoisomerase 2␤ (Top 2␤) [1,2,3,4,5,6,7]. This enzyme has been mapped to the 5 end of transcriptionally active genes in several experimental systems [8,9,10]. Many restriction enzymes generate ss incisions at imperfect recognition motifs [19,20], that are mistaken for endoge-

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Conclusion