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Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA

An Addendum to this article was published on 19 July 2019

Abstract

Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed ribose-seq, a method for capturing unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly synthesized leading strand of nuclear DNA and were present upstream of (G+C)-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA.

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Figure 1: Ribose-seq method for mapping rNMPs in genomic DNA.
Figure 2: Distribution of rNMP incorporation in the S. cerevisiae genome.
Figure 3: Identity and sequence contexts of rNMP incorporation in S. cerevisiae genome.
Figure 4: Hotspots of rNMP incorporation in S. cerevisiae mitochondrial DNA, rDNA repeat and Ty1.

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Acknowledgements

We thank N.V. Hud and L.D. Williams for support with urea-PAGE gels and for advice on this study and the manuscript; M. Goodman, B. Weiss and I.K. Jordan for suggestions on this study and the manuscript and assistance with data analysis; S. Garrey for AtRNL and Tpt1 protein purification; C. Cox for sequencing; Y. Shen for assistance with statistical analysis; A. Gombolay and L. Shetty for technical help; and all members of the Storici laboratory for advice in the course of the study. This research was supported by US National Science Foundation award number MCB-1021763 (to F.S.), Georgia Research Alliance award number R9028 (to F.S.), an American Cancer Society Research Scholar Grant (to J.R.H.), a Damon Runyon-Rachleff Innovation Award from the Damon Runyon Cancer Research Foundation (to J.R.H.) and the University of Colorado Golfers Against Cancer (to J.R.H.).

Author information

Authors and Affiliations

Authors

Contributions

K.D.K. conducted all of the experiments for in vitro biochemical assays and ribose-seq library construction, and most of the experiments for yeast in vivo DSB repair assay with oligonucleotides, with assistance from S.B. J.R.H. conducted the sequencing analysis. F.S., together with K.D.K. and J.R.H., designed experiments, assisted in data analysis and wrote the manuscript.

Corresponding authors

Correspondence to Jay R Hesselberth or Francesca Storici.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mechanism of alkaline cleavage of ribonucleotides in DNA

The ribonucleoside embedded in double-stranded DNA is in red. During alkaline treatment, DNA strands are denatured, and cleavage occurs at the rNMP site, generating a 2′,3′-cyclic phosphate end and an opposite 5′-hydroxyl end. The 2′,3′-cyclic phosphate is in equilibrium with 2′-phosphate and 3′-phosphate forms. Boxes in black indicate the 2′,3′-cyclic phosphate and 2′-phosphate DNA termini, which are substrates of AtRNL.

Supplementary Figure 2 3′ base bias for AtRNL ligation

Hot 5′-radiolabeled 30-nt DNA oligo with a single rNMP (either A, G, U, or C) in the 22nd position was mixed with cold equimolar 30-nt DNA oligos with rNMPs of 3 other bases in the 22nd positions. 5′-radiolabel is indicated by ‘P’ in purple. The mixture was treated with 0.3M NaOH for 2 hr at 55 °C and neutralized. 100 nM of alkali-cleaved products (25 nM of each base) were then incubated with 1 µM AtRNL in appropriate buffer (see Methods) for 1 hr at 30 °C. The resulting products were treated with T5 exonuclease for 2 hr at 37 °C. Aliquots were withdrawn after appropriate steps and quenched. The products were analyzed by urea-PAGE. The circular 22-mer migrates faster than the unligated, linear 22-mer. Only circular products were resistant to T5 exonuclease while all linear substrates/products were degraded. Median percentages of circular 22-mer formation from four independent reactions are displayed. See Supplementary Table 1 for more statistics. First left lane, ss DNA ladder. No 3′ base bias was observed for AtRNL ligation (see Supplementary Table 1). Self-ligation was preferred to dimerization with a shorter 22-nt substrate; however, with the shorter substrate, lower levels of linear dimers, which are not resistant to T5 exonuclease, and circular dimers were observed. Increasing the length of the ss DNA substrate from 22 nt to 32 nt eliminated dimerization (Fig. 1a).

Supplementary Figure 3 Ribose-seq library from genomic DNA of S. cerevisiae rnh201Δ (KK-100) cells

Appropriate PCR products were analyzed by PAGE. ‘P’ indicates primers-only. No amplification product was observed when either (a) AtRNL ligation step or (b) alkali treatment was omitted. Tpt1 denotes the step of 2’-phosphate removal at the ligation junction in Figure 1a. First left lane, ds DNA ladder.

Supplementary Figure 4 Bypass of a single rNMP by Phusion DNA polymerase

5′-radiolabeled 30-nt primer, ByPrim (Supplementary Table 8), was annealed to the 46-nt template oligo containing either rCMP (ByTemp.rC) or rUMP (ByTemp.rU) in the 8th position. 100 nM of annealed substrate was incubated with 0.2 units of Phusion High-Fidelity DNA Polymerase (NEB) and 2 mM dNTPs in appropriate buffer (see Methods) for 30 sec at 72 °C. The reactions were quenched and analyzed by urea-PAGE. Median bypass probabilities from four independent reactions are shown. See Supplementary Table 5 for more statistics. First left lane, ss DNA ladder. The primer extension assay showed no significant difference between bypass efficiency over rUMP and rCMP by Phusion DNA polymerase (Supplementary Table 5).

Supplementary Figure 5 Normalized frequency of nucleotides surrounding the rNMP sites

Normalized frequency of nucleotides relative to (a) nuclear and (b) mitochondrial mapped positions of sequences from ribose-seq library, PCR-amplified with EconoTaq DNA Polymerase (Lucigen), of genomic DNA from S. cerevisiae rnh201Δ (KK-100) cells. Position 0 corresponds to the rNMP. Negative and positive numbers (from -10 to -1 and 1 to 10) correspond to upstream and downstream positions from the rNMP, respectively. Frequencies were normalized to either nuclear or mitochondrial genomic mononucleotide frequencies. Normalized frequency of nucleotides relative to (c) nuclear and (d) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh201Δ (KK-30) cells. Normalized frequency of nucleotides relative to (e) nuclear and (f) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ (KK-174) cells. Normalized frequency of nucleotides relative to (g) nuclear and (h) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ (KK-125) cells. Normalized frequency of nucleotides relative to (i) nuclear and (j) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ ung1Δ (KK-164) cells. Normalized frequency of nucleotides relative to (k) nuclear and (l) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae pol2-M644G rnh201Δ (KK-170) cells. Normalized frequency of nucleotides relative to (m) nuclear and (n) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae pol3-5DV rnh201Δ (KK-120) cells.

Supplementary Figure 6 Zoom-out of normalized frequency of nucleotides surrounding the rNMP sites

Normalized frequency of nucleotides relative to (a) nuclear and (b) mitochondrial mapped positions of sequences from ribose-seq library, PCR-amplified with EconoTaq DNA Polymerase (Lucigen), of genomic DNA from S. cerevisiae rnh201Δ (KK-100) cells. Position 0 corresponds to the rNMP. Negative and positive numbers (from -100 to -1 and 1 to 100) correspond to upstream and downstream positions from the rNMP, respectively. Frequencies were normalized to either nuclear or mitochondrial genomic mononucleotide frequencies. Normalized frequency of nucleotides relative to (c) nuclear and (d) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh201Δ (KK-30) cells. Normalized frequency of nucleotides relative to (e) nuclear and (f) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ (KK-174) cells. Normalized frequency of nucleotides relative to (g) nuclear and (h) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ (KK-125) cells. Normalized frequency of nucleotides relative to (i) nuclear and (j) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ ung1Δ (KK-164) cells. Normalized frequency of nucleotides relative to (k) nuclear and (l) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae pol2-M644G rnh201Δ (KK-170) cells. Normalized frequency of nucleotides relative to (m) nuclear and (n) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae pol2-4 rnh201Δ (KK-107) cells. Normalized frequency of nucleotides relative to (o) nuclear and (p) mitochondrial mapped positions of sequences from ribose-seq library of genomic DNA from S. cerevisiae pol3-5DV rnh201Δ (KK-120) cells.

Supplementary Figure 7 Targeting of rGMP and rUMP by RNase H2 and uracil DNA N-glycosylase during DSB repair in S. cerevisiae cells

a, Diagram and sequence of the chromosomal leu2 region targeted by DNA-control LEU2.D, rGMP-containing LEU2.rG, dUMP-containing LEU2.dU, and rUMP-containing LEU2.rU oligos (Supplementary Table 8). StuI recognition sequence is underlined in turquoise. Position of either rGMP, dUMP, or rUMP was selected so that it is about 4-5 nt upstream of the G-T mispair. Both RNase H2-initiated excision repair (RER) and base excision repair (BER) remove a short ss DNA region downstream of the damage during the repair13,27. b, The oligos were transformed to either RNase H2- and uracil DNA N-glycosylase-proficient wild-type (WT; FRO-767,768), RNase H2-deficient (rnh201; FRO-984,985), or DNA N-glycosylase-deficient (ung1; KK-158,159) S. cerevisiae cells (see Supplementary Table 2). Median percentages of StuI-cut Leu+ transformants from four independent transformations are shown with ranges as bars. For each transformation, 20 Leu+ transformants were selected for analysis. Mann-Whitney U-test was implemented for statistical analysis against the WT. P values of less than 0.05 are marked as asterisk. See Supplementary Table 6 for more statistics.

Supplementary Figure 8 Normalized frequency of nucleotides surrounding the rNMP sites on leading and lagging strands

Normalized frequency of nucleotides relative to mapped positions of sequences in (a) leading and (b) lagging strands from ribose-seq library of genomic DNA from S. cerevisiae rnh1Δ rnh201Δ (KK-174) cells. Position 0 corresponds to the rNMP. Negative and positive numbers (from -10 to -1 and 1 to 10) correspond to upstream and downstream positions from the rNMP, respectively. ARSs with Trep of no longer than 25 min were selected with flanking size of 10 kb. Frequencies were normalized to genomic mononucleotide frequencies of either leading or lagging strand of the selected ARSs and flanking size. Normalized frequency of nucleotides relative to mapped positions of sequences in (c) leading and (d) lagging strands from ribose-seq library of genomic DNA from S. cerevisiae pol3-5DV rnh201Δ (KK-120) cells.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1–8 (PDF 4491 kb)

Supplementary Data

Results of peak calling analysis of all ribose-seq libraries in this study. (XLS 135 kb)

Supplementary Software

Modmap software version 1.1 includes the pipeline for sequencing analysis (using bowtie, samtools and bedtools) and a series of bash, Python and R scripts for analyzing data and generating plots. The software is available here as a ZIP file and is maintained at http://github.com/hesselberthlab/modmap/. (ZIP 360 kb)

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Koh, K., Balachander, S., Hesselberth, J. et al. Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA. Nat Methods 12, 251–257 (2015). https://doi.org/10.1038/nmeth.3259

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