High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase

Nucleic Acids Res. 2020 May 7;48(8):4274-4297. doi: 10.1093/nar/gkaa103.


Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Damage
  • DNA Topoisomerases, Type I / metabolism*
  • Deoxyribonucleotides / metabolism
  • Genome, Fungal
  • Genomic Instability
  • Mutation
  • Ribonuclease H / genetics
  • Ribonucleases / genetics
  • Ribonucleotide Reductases / genetics*
  • Ribonucleotides / metabolism*
  • S Phase Cell Cycle Checkpoints
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Deletion


  • Deoxyribonucleotides
  • Ribonucleotides
  • Saccharomyces cerevisiae Proteins
  • Ribonucleotide Reductases
  • Rnr1 protein, S cerevisiae
  • Ribonucleases
  • Rnh201 protein, S cerevisiae
  • Ribonuclease H
  • ribonuclease HI
  • TOP1 protein, S cerevisiae
  • DNA Topoisomerases, Type I