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

Nature Structural & Molecular Biology volume 22, pages 291–297 (2015)Cite this article

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Abstract

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.

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Figure 1: Ribonucleotide incorporation in vitro by variants of Pols α and δ.
Figure 2: Strand-specific probing for ribonucleotides in nascent lagging-strand genomic DNA.
Figure 3: Lack of Top1-initiated ribonucleotide removal in pol1-L868M rnh201Δ and pol3-L612M rnh201Δ strains.
Figure 4: RNase H2 is dispensable for maintaining genome integrity in strains with increased capacity to incorporate ribonucleotides into lagging-strand DNA.
Figure 5: A model depicting three possibilities for strand-specific consequences of unrepaired ribonucleotides in the genomes of RER-defective yeast strains.

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Acknowledgements

We thank K. Bebenek, C. Orebaugh and S. Williams for helpful comments on the manuscript and all members of the Kunkel laboratory for thoughtful discussions. We acknowledge the US National Institute of Environmental Health Sciences (NIEHS) Molecular Genetics Core Facility for sequence analysis of 5-FOA–resistant mutants and the NIEHS Flow Cytometry Center for fluorescence-activated cell-sorting analysis. This work was supported by project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the US National Institutes of Health (NIH), NIEHS, by the Swedish Cancer Society to A.C. and by US NIH grant GM032431 to P.M.B.

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Authors and Affiliations

  1. Department of Health and Human Services, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Jessica S Williams, Anders R Clausen, Scott A Lujan, Alan B Clark & Thomas A Kunkel

  2. Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden

    Lisette Marjavaara & Andrei Chabes

  3. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA

    Peter M Burgers

  4. Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden

    Andrei Chabes

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Contributions

J.S.W., A.R.C., S.A.L., L.M., A.B.C. and A.C. designed and performed experiments. P.M.B. provided reagents. All authors were involved in data analysis. J.S.W. and T.A.K. wrote the manuscript, with input from all authors.

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Correspondence to Thomas A Kunkel.

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Integrated supplementary information

Supplementary Figure 1 Probing for ribonucleotides in nascent leading-strand DNA.

(a) The annealing locations of radiolabeled leading strand-specific probes on the URA3 reporter in OR1 or OR2. (b) Detection of alkali-sensitive sites in nascent leading strand DNA synthesized by Pol ɛ was performed as described13. Smaller DNA fragments observed for the α-LM rnh201Δ and δ-LM rnh201Δ mutants when using probes that anneal to the nascent leading strand (lanes 4, 6, 12 and 14) may be related to the close proximity of URA3 to ARS306 (1.6 kb). These alkali-sensitive sites may arise during ribonucleotide incorporation by Pols α or δ into the nascent lagging strand during bidirectional synthesis proceeding from this origin in the opposite direction (to the left of the origin in panel a). In addition, these small fragments that hybridize to the ‘leading strand’ probe may be generated by synthesis performed by L868M Pol α or L612M Pol δ as they replicate from the adjacent ARS307 origin. The same explanation applies for small DNA fragments observed for the ɛ-MG rnh201Δ mutant in Figure 2 (main text) when using probes that anneal to the nascent lagging strand (lanes 8 and 16). (c) The average fraction of alkali-sensitive fragments along the membrane was determined by quantifying the radioactive signal using data from in panel b (for both Probes A and B) from two independent experiments.

Supplementary Figure 2 Workflow for calculating fractional replication-strand bias from HydEn-seq end counts without background subtraction or internal standards.

Each set of arrows indicates the application of the equation shown to the left. Gray data points represent bins of 20 base pairs (bp). Labels above each graph are relate the variables in the equations to the values for which they are color-coded. Solid lines are moving averages over 25 bins (500 bp). (a) HydEn-seq end counts for reads mapping to the forward (black) and reverse (orange) strands from RNase H2-deficient pol1-L868M (α-LM; left), pol2-M644G (ɛ-MG; middle), and pol3-L612M (δ-LM; right) strains. (b) The observed strand bias, expressed as a log-ratio between forward- and reverse-strand end counts. (c) An approximation of the true replication strand bias log-ratio. Extra-replicative contributions were removed by comparing strains with opposite biases. The greater the biases in the chosen strains, the closer the approximation. (d) The fraction of replication events in which the bottom strand originates as the nascent lagging strand.

Supplementary Figure 3 Alkali-sensitive Okazaki fragment–sized DNA is not observed in the α-LM rnh201Δ or δ-LM rnh201D strains.

Purified genomic DNA was subjected to alkaline hydrolysis, alkaline-agarose electrophoresis and stained with SYBR® Gold Nucleic Acid Gel Stain, a sensitive fluorescent stain that can be used for detection of single strand DNA. The fraction of alkali-sensitive fragments was calculated by dividing the fluorescence intensity (arbitrary units) at each position along the gel by the total intensity for each lane. The experiment was performed in duplicate, and a representative gel image and quantitation is displayed. We note that Okazaki fragment-sized DNA (e.g. 150-300 base pair) fragments were not observed among the products of the alkaline hydrolysis of genomic DNA from the α-LM rnh201Δ or δ-LM rnh201Δ strains.

Supplementary Figure 4 Asymmetric phenotypes are associated with unrepaired ribonucleotides incorporated into nascent leading- versus lagging-strand DNA.

(a) Cell cycle progression is not affected by loss of RNH201 in the pol1-LM or the pol3-LM mutator strains with an increased number of unrepaired ribonucleotides in the nascent lagging strand. Cells were grown to mid-log phase at 30°C and processed for flow cytometry as described in13. The experiment was performed in duplicate and data are displayed as the mean % ± standard error. (b) The increase in total dNTP abundance conferred by loss of RNH201 is not significantly enhanced in the pol1-LM or the pol3-LM lagging strand polymerase mutator strains. Data for total dNTP abundance (dCTP, dATP, dTTP and dGTP) is displayed as the mean ± standard error. Each strain genotype was independently analyzed twice.

Supplementary Figure 5 URA3 mutation spectra for pol1 -L868M rnh201 Δ and pol3-L612M rnh201 Δ.

The coding strand of the 804 base pair URA3 open reading frame is shown. The sequence changes observed in independent ura3 mutants are depicted above the coding sequence for pol1-L868M rnh201∆ in red and below the coding sequence for pol3-L612M rnh201∆ in blue. Letters indicate single base substitutions, open triangles indicate single base deletions, and short lines above or below the coding sequence indicate multibase base deletions of between 2 and 5 base pairs. All strains had the URA3 reporter in OR2.

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Supplementary Figures 1–5 and Supplementary Tables 1–3 (PDF 5273 kb)

Supplementary Data Set 1

Original gel images for Figures 1–3 (PDF 28958 kb)

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Williams, J., Clausen, A., Lujan, S. et al. Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific. Nat Struct Mol Biol 22, 291–297 (2015). https://doi.org/10.1038/nsmb.2989

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