Ribonucleotides and Transcription-Associated Mutagenesis in Yeast

Abstract

High levels of transcription stimulate mutation rates in microorganisms and this occurs primarily through an enhanced accumulation of DNA damage. The major source of transcription-associated damage in yeast is topoisomerase I (Top1), an enzyme that removes torsional stress that accumulates when DNA strands are separated. Top1 relieves torsional stress by nicking and re-sealing one DNA strand, and some Top1-dependent mutations are due to trapping and processing of the covalent cleavage intermediate. Most, however, reflect enzyme incision at ribonucleotides, which are the most abundant non-canonical component of DNA. In either case, Top1 generates a distinctive mutation signature comprised of short deletions in tandem repeats; in the specific case of ribonucleotide-initiated events, mutations reflect sequential cleavage by the enzyme. Top1-dependent mutations do not require highly activated transcription, but their levels are greatly increased by transcription, which partially reflects an interaction of Top1 with RNA polymerase. Recent studies have demonstrated that Top1-dependent mutations exhibit a strand bias, with the nature of the bias differing depending on the transcriptional status of the underlying DNA. Under low-transcription conditions, most Top1-dependent mutations arise in the context of replication and reflect incision at ribonucleotides incorporated during leading-strand synthesis. Under high-transcription conditions, most Top1-dependent events arise when the enzyme cleaves the non-transcribed strand of DNA. In addition to increasing genetic instability in growing cells, Top1 activity in transcriptionally active regions may be a source of mutations in quiescent cells.

Both R-loop removal and ribonucleotide excision repair activities of RNase H2 contribute substantially to chromosome stability

“…For example, 2–5 bp deletions accumulate at low complexity regions in RNase H2 mutants [6]. This phenotype stems from a defect in normal RER initiation, which then affords an opportunity for mutagenic processing by topoisomerase 1 at regions containing rNMPs [7–11].…”

RNases H1 and H2: guardians of the stability of the nuclear genome when supply of dNTPs is limiting for DNA synthesis

“…This could produce a non-ligatable, single-strand nick that compromises genome integrity [for a review, see e.g. (Cho and Jinks-Robertson 2017, Cho and Jinks-Robertson 2018, Williams, et al 2016]. For instance, in budding yeast, Top1-mediated incisions within short tandem repeats at sites of unrepaired single genomic ribonucleotides (i.e.…”

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair

“…In this case, rNMPs could physically influence RNA transcription to varying degrees, depending on whether the rNMP resides in the transcribed or the non-transcribed strand, whether a gene is expressed at high or low levels, and where within the gene the rNMP is located. Transcription-associated mutagenesis can indeed be linked to unrepaired ribonucleotides (Cho & Jinks-Robertson, 2017). The characteristic two-to five-basepair slippage mutations generated by Top1 at unrepaired rNMPs are elevated within actively transcribed hotspot regions (Takahashi et al, 2011).…”