Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase I

Abstract

The ribonuclease (RNase) H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides [ribonucleoside monophosphates (rNMPs)] from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.

Figure 1. Mutagenesis in the CAN1 forward mutation assay

(A and B) show partial mutation spectra of Can-R mutants (complete spectra are in Figure S1). Nucleotides are numbered beginning with the ATG start codon. Base substitutions and indels are in red below and above the sequence, respectively; lengths of red bars correspond to deletion sizes. Sequences transplanted into the lys2ΔA746NR assay are highlighted in yellow. C presents rates of individual mutation types at CAN1. N, number of mutants sequenced; indel, insertion/deletion; BS, base substitution. 95% confidence intervals are in parentheses below total rates.

Figure 2. Rates of mutation types in lys2 frameshift reversion assays

Gray bars correspond to 2-bp deletions at the introduced hotspot. White and black bars represent 1-bp indels and all other classes of mutations, respectively.

Figure 3. Top1 cleavage assays

(A and B) illustrate the mechanism of Top1 cleavage at a scissile dNMP versus rNMP, respectively. (C and D) show cleavage of (AT)₂ and (TC)₃ hotspot fragments, respectively, by human Top1. In the fragment sequences, relevant dinucleotide repeats are highlighted in gray and ribo-substituted nucleotides are in bold italics. Transcribed and nontranscribed strands are designated TS and NTS, respectively; the TS strand was radioactively labeled at either the 3′ or 5′ end as indicated. Labeled arrows indicate positions of Top1 cleavage. Time points for the salt reversal experiments are in min.

Figure 4. Model for Top1-initiated deletions

The (AT)₂ hotspot sequence is shown; the top strand is the nontranscribed strand and the dinucleotide repeat highlighted in gray. The cyclic 2′,3′ phosphate formed by Top1 cleavage at rUMP is indicated by a triangle.