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) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. 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.
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.
Jessica S
JS
Williams
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.
Anders R
AR
Clausen
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.
Scott A
0000000324008325
SA
Lujan
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
Lisette
L
Marjavaara
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.
Alan B
AB
Clark
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
Peter M
PM
Burgers
1] Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden. [2] Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
Andrei
0000000317088259
A
Chabes
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA.
Thomas A
TA
Kunkel
D004247:DNA / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism / Q000502:physiology; D004260:DNA Repair; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism / Q000502:physiology*; D012265:Ribonucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*
D012265:Ribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004257:DNA Polymerase II; D004264:DNA Topoisomerases, Type I