Topoisomerase 1-dependent deletions initiated by incision at ribonucleotides are biased to the non-transcribed strand of a highly activated reporter

Abstract

DNA polymerases incorporate ribonucleoside monophosphates (rNMPs) into genomic DNA at a low level and such rNMPs are efficiently removed in an error-free manner by ribonuclease (RNase) H2. In the absence of RNase H2 in budding yeast, persistent rNMPs give rise to short deletions via a mutagenic process initiated by Topoisomerase 1 (Top1). We examined the activity of a 2-bp, rNMP-dependent deletion hotspot [the (TG)2 hotspot] when on the transcribed or non-transcribed strand (TS or NTS, respectively) of a reporter placed in both orientations near a strong origin of replication. Under low-transcription conditions, hotspot activity depended on whether the (TG)2 sequence was part of the newly synthesized leading or lagging strand of replication. In agreement with an earlier study, deletions occurred at a much higher rate when (TG)2 was on the nascent leading strand. Under high-transcription conditions, however, hotspot activity was not dependent on replication direction, but rather on whether the (TG)2 sequence was on the TS or NTS of the reporter. Deletion rates were several orders of magnitude higher when (TG)2 was on the NTS. These results highlight the complex interplay between replication and transcription in regulating Top1-dependent genetic instability.

The Balancing Act of Ribonucleotides in DNA

“…S. cerevisiae rnh1Δrnh201Δ strains are viable but require Top1 activity to prevent R-loop formation during transcription, most notably in rRNA genes[67, 68]. Highly transcribed genes in a wildtype RNH1 RER-defective strain also exhibit 2-5 bp deletions on the non-transcribed strand(NTS), regardless of direction of replication[69]. In this instance, even overexpression of RNase H1 fails to eliminate the deletions.…”

The role of RNase H2 in processing ribonucleotides incorporated during DNA replication

“…This nick cannot be sealed by ligases, but it can be reversed by Top1 [15, 16]. However, when nicking occurs in a short tandemly repeated sequence, a second Top1-dependent cleavage can occur and result in loss of one of the repeated sequences [17]. Thus, deletion of the gene encoding the catalytic subunit of yeast RNase H2 ( RNH201 ) causes a high rate of 2–5 base pair deletions in short tandem-repeat sequences [18, 19].…”

Ribonucleotides and Transcription-Associated Mutagenesis in Yeast

“…When a given hotspot is strongly affected by the direction of DNA replication, the strand cleaved by Top1 can be deduced. At one specific hotspot, this logic was used to identify the (TG) 2 -containing, rather than the complementary (CA) 2 -containing, strand as the strand targeted by Top1 [72]. Insertion of pLYS2 -driven (TG) 2 reporter next to a well-defined origin of replication confirmed the strong replication-orientation bias for 2-bp deletions at this particular hotspot.…”

“…That is, deletions accumulated primarily when Top1 cleaves the NTS, regardless of whether this strand is generated during leading- or lagging-strand synthesis (Figure 3, boxes with solid black lines). Thus, in contrast to low-transcription conditions where only those ribonucleotides incorporated by Pol ε are highly mutagenic [11], Pol δ can be the major source of deletion-causing rNMPs under high-transcription conditions [72]. …”

“…One possibility is that nicked/gapped intermediates arising on the TS are subject to transcription-coupled nucleotide excision repair (NER), which would preclude their maturation into a deletion event. Elimination of NER, however, does not alter the NTS bias [72]. A second possibility is that Top1 is only able to cleave the NTS, perhaps because the TS strand is shielded as part of an RNA:DNA hybrid.…”