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

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.

Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage

“…Single-stranded DNA is exposed following fission yeast condensin inactivation [61], again dependent on active transcription [23]. Together with the observation of naturally occurring single-stranded DNA breaks at promotors of active genes and transcription-dependent promoter decompaction [62,63], this highlights the challenge to genome stability that arises from DNA unwinding and topological strain associated with gene transcription. To understand how condensin conveys its protective effect, it will be important to map locations of preferential condensin binding in the interphase nucleus, as well as the locations of the fragile sites where DNA breaks in its absence.…”

Evolution of resistance to Irinotecan in cancer cells involves generation of topoisomerase-guided mutations in non-coding genome that reduce the chances of DNA breaks

“…This work also illuminates novel aspects of function of Top1. Though it was reported that Top1 associates with active RNA polymerase to relieve DNA supercoils generated in the process of transcription (41)(42)(43), our data suggests that Top1 can also functions in a transcription-independent relief of supercoils since a majority of mutations was seen in heterochromatin (H3K9me3 and H3K27me3 patterns, see Fig. S2).…”

KDM4-dependent DNA breaks at active promoters facilitate +1 nucleosome eviction

“…7) shifted back the stability of the H3K4me3 nucleosomes to those of the control, showing that topoisomerase II was in place and potentially active, but rendered inactive in the absence of KDM4 or without the enzymatic activities involved. The decrease of nicks upon KO induction was also analyzed in an experiment when the overall nick levels were studied by a reverse Southwestern (rSW) blot procedure 55 , as shown in Suppl. Fig.…”