Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific

Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA-damage phenotypes. Here we show that these phenotypes are incurred by a high level of ribonucleotides incorporated by a leading strand-replicase variant, DNA polymerase (Pol) epsilon, but not by orthologous variants of the lagging-strand replicases, Pols alpha or delta. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol epsilon but not by Pols alpha or delta. Several explanations for this asymmetry are considered, including the idea that Top1 incision at ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension.