Two distinct mechanisms of Topoisomerase 1-dependent mutagenesis in yeast

Abstract

Topoisomerase 1 (Top1) resolves transcription-associated supercoils by generating transient single-strand breaks in DNA. Top1 activity in yeast is a major source of transcription-associated mutagenesis, generating a distinctive mutation signature characterized by deletions in short, tandem repeats. A similar signature is associated with the persistence of ribonucleoside monophosphates (rNMPs) in DNA, and it also depends on Top1 activity. There is only partial overlap, however, between Top1-dependent deletion hotspots identified in highly transcribed DNA and those associated with rNMPs, suggesting the existence of both rNMP-dependent and rNMP-independent events. Here, we present genetic studies confirming that there are two distinct types of hotspots. Data suggest a novel model in which rNMP-dependent hotspots are generated by sequential Top1 reactions and are consistent with rNMP-independent hotspots reflecting processing of a trapped Top1 cleavage complex.

Fig. 1. End-processing model for Top1-dependent deletions

Light grey boxes indicate the relevant dinucleotide repeat. Yellow arrows designate Top1 cleavage. See section 1 for details.

Fig. 2. 2–5 bp deletions at CAN1

A. Rate of 2–5 bp deletions. The rate under low-transcription in an rnh201Δ top1Δ background is not shown because it was determined in a different strain background [14]. Error bars show 95% confidence intervals. B. Locations of 2–5 bp deletions. Only a part of CAN1 ORF is shown. Grey bars over the sequence and red bars below the sequence represent deletion events observed in the WT and rnh201Δ background, respectively (Table S2). The length of the bars corresponds to the size of deletion. Light grey boxes highlight all dinucleotide repeats. The (AT)₂, (TC)₃, and (AG)₄ hotspot-containing sequences transplanted into the lys2ΔA746,NR reversion window are highlighted with yellow boxes.

Fig. 3. Reversion rates of lys2ΔA746NR alleles

The LYS2 and TET promoters were used for low- and high-transcription conditions, respectively. Grey bars represent 2-bp deletions (Table S2). White bars represent other events. Low-transcription data and WT high-transcription data were previously reported [8, 14]. The 95% confidence intervals are indicated.

Fig. 4. Effect of Pol2-M644L and Top1-T722A expression on 2-bp deletions under high-transcription conditions

A. Effect of Pol2-M644L on 2-bp deletion rates in an rnh201Δ background (Table S2). B. Effect of Top1-T722A expression on 2-bp deletion frequencies in top1Δ rnh201Δ backgrounds (Table S3). Frequencies observed with an empty vector control or WT Top1 expression also are shown. Error bars represent 95% confidence intervals of rates (A) and median frequencies (B).

Fig. 5. Reversion rates of lys2ΔBgl and lys2ΔA746 alleles

A. The lys2ΔBgl allele was under control of the TET promoter. Low-and high-transcription conditions were created by growth in the presence or absence of doxycycline, respectively. Dark grey bars correspond to 4-bp deletions at (AGCT)₂ and light grey bars to 1-bp deletions at the 6A run (Table S4). B. The LYS2 and TET promoters were used for low- and high- transcription conditions, respectively. Black bars correspond to 2-bp deletions at the 6A run and white bars to other events (Table S4). The 95% confidence interval of the total Lys⁺ rate is indicated.

Fig. 6. Sequential Top1 cleavage model for rNMP-associated deletions

Light grey boxes indicate the relevant dinucleotide repeat. Yellow arrows designate Top1 cleavage. See section 4 for details.