Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

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Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′ DNA end–mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair–deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5′ DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

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Figure 1: Mapping ribonucleotides by HydEn-seq.
Figure 2: Strand-specific ribonucleotide mapping of chromosome 10.
Figure 3: Genome-wide replication origins located by HydEn-seq.
Figure 4: Distribution of ribonucleotides near origins in RER-deficient strains.
Figure 5: Ribonucleotide base identity.
Figure 6: Meta-analysis of ribonucleotides at the nucleosome dyad.
Figure 7: HydEn-seq maps of mitochondrial DNA.

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We thank M. Young and M. Longley for helpful comments on the manuscript. This work was supported by the Division of Intramural Research of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences (project Z01 ES065070 to T.A.K.) and by NIH grant 2R01GM052319-16A1 to P.A.M.

Author information

A.R.C., D.J.S. and T.A.K. designed the experiments, A.R.C., C.D.O., J.S.W., M.F.C. and E.P.M. performed the experiments, A.R.C., S.A.L., A.B.B., D.C.F., P.A.M. and T.A.K. analyzed the data, T.A.K. wrote the manuscript, and all authors edited the manuscript.

Correspondence to Thomas A Kunkel.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Representative 5′ DNA end reads for the previously shown region of chromosome 10.

The region shown matches that of Fig. 2a, bottom panel. All strains are RER-defective (rnh201Δ). The numerical values on the right side of each tract indicate the maximum number of reads in that tract.

Supplementary Figure 2 Heat maps for RNH201 libraries.

Supplementary Figure 3 Evolutionary conservation of the three major eukaryotic nuclear replicases.

(a) Expanded from. Motif A is depicted for the three major eukaryotic nuclear replicases, with residues relevant to this study highlighted in bold and located immediately adjacent to the invariant tyrosine that acts as a steric gate to reduce ribonucleotide incorporation. The number of sequences found for each clade is are shown in parentheses. (b) RNase H2 subunit A catalytic motifs (Chon, H. et al. RNase H2 roles in genome integrity revealed by unlinking its activities. Nucleic Acids Research 41, 3130-3143 (2013)) are similar across the three kingdoms of life. (c) A phylogenetic tree shows RNase H2 subunit A conservation across all major taxa (2396 sequences; less than 1 false positive expected; see Supplementary Table 6).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Tables 1–4 (PDF 1511 kb)

Supplementary Table 5

List of confirmed and new origins observed by HydEn-seq (XLSX 33 kb)

Supplementary Table 6

RNase H2 subunit A BLAST parameters and hits (XLSX 833 kb)

Supplementary Data Set 1

Uncropped gel image from Figure 1b (PDF 1949 kb)

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Clausen, A., Lujan, S., Burkholder, A. et al. Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22, 185–191 (2015) doi:10.1038/nsmb.2957

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