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 resealing 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 noncanonical component of DNA. In either case, Top1 generates a distinctive mutation signature composed 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.

Keywords: Topoisomerase 1; mutagenesis; ribonucleotides; transcription.

Figure 1

Error-free and error-prone removal of ribonucleotides from DNA. In the error-free pathway (gray arrows on the left side), RNase H2 incises 5’ of the ribonucleotide (red rectangle) to generate a 3’-OH and 5’-phosphate (P) end. Following stand displacement synthesis by Pol δ or Pol ε (blue pentagon) the 5’ flap is removed by Rad27 or Exo1 and the remaining nick is sealed by ligase. Black lines indicate DNA and arrowheads designate 3’-OH ends; dotted lines indicate new DNA synthesis. As an alternative to strand displacement after RNase H2 incision, abortive ligation generates a toxic 5’-AMP attached to the ribose, which can be reversed by aprataxin. The error-prone pathway of ribonucleotide removal (yellow arrows on the right side) generates deletions via sequential Top1 (yellow oval) cleavage. In contrast to RNase H2 incision, Top1 cleaves 3’ of the ribonucleotide to generate a 5’-OH and a covalent linkage of the enzyme to a 3’-phosphate. Nucleophilic attack of the Top1-DNA covalent bond by the 2’-OH of the ribonucleotide releases Top1, leaving behind a nick flanked by a 5’-OH and 2’,3’-cyclic phosphate (red triangle attached to the red rectangle). Top1 either facilitates ligation of the ends through the reverse reaction or cleaves DNA a second time. The second cleavage generates a short oligo that is released, resulting in trapping of the Top1cc. Spontaneous realignment of complementary strands brings the 5’-OH and Top1cc into close proximity to facilitate Top1-mediated ligation. Removal of 5’-OH by Srs2 and Exo1 prevents Top1-mediated ligation.

Figure 2

Ribonucleotide-dependent deletions during replication and transcription in the absence of RNase H2. Black lines are DNA and arrowheads designate 3’-OH ends. Dotted lines are DNA synthesized during replication and solid red lines are RNA primers for DNA synthesis. Red and blue pentagons correspond to Pol ε and Pol δ, respectively. “R” corresponds to ribonucleotides; those inserted by Pol ε and Pol δ are red and blue, respectively. The red line trailing RNAP is the RNA transcript. Top1 (yellow oval) is tethered to components of the replication (CMG helicase in green) and transcription machineries (purple oval moving in the direction of the purple arrow) to relieve supercoils formed during each. During replication, supercoils form on the leading strand of replication and Top1 incision generates deletions primarily at ribonucleotides incorporated by Pol ε. During transcription, deletions reflect incision at ribonucleotides located on the NTS, which can be inserted by either Pol ε or Pol δ.

Figure 3

Strand bias of Top1 cleavage at ribonucleotides during replication and transcription. The experimental set-up used to determine strand specificities of Top1 cleavage in the absence of RNase H2 is illustrated. The white arrow indicates the NTS of a hotspot-containing reporter. The yellow box represents a Top1 cleavage site and the white box corresponds to the non-scissile, complementary strand. The red and black lines correspond to DNA synthesized by Pol ε and Pol δ, respectively, and the ribonucleotide (R) incorporated by each is similarly colored. A small fragment containing yellow/white boxes was inverted within the reporter to switch the strand on which the cleavage site resides; this switches the cleavage site between the TS-NTS and between the strands synthesized by Pol ε and Pol δ. The direction of replication was reversed by inverting the entire reporter, thereby switching the strands synthesized by Pol ε and Pol δ. Under low-transcription conditions, deletions were observed at a high level only in the two configurations marked with a red dotted box. The common feature is that the yellow box contains a red R incorporated by Pol ε. Under high-transcription conditions, deletions accumulated only in configurations marked with black box. In both cases, the yellow box is on NTS.

Figure 4

Encounters with RNAP promote deletions only when Top1 cleaves the NTS. Black lines correspond to DNA and arrowheads indicate 3’ ends. A deletion hotspot with three repeat units (boxes) is shown in each panel; inversion of the hotspot moves the cleavage sites from the NTS to the TS in the panels on the left and right, respectively. The Top1cc (yellow oval) is trapped at the end of the red repeat; the repeat within the gap created by sequential Top1 cleavage is in gray. The 5’-OH that borders the gap was generated by the first Top1 incision at a ribonucleotide. The blue arrow depicts the direction of RNAP (blue oval) movement and the trailing red line is the nascent RNA. When the Top1cc is trapped on the NTS, the approaching RNAP pushes it (yellow arrow) and the attached repeat towards the 5’-OH to facilitate ligation. When the Top1cc is trapped on the TS, an advancing RNAP pulls the 5’-OH away from the Top1cc, preventing Top1-mediated ligation.