Abstract
Ribonucleotides are frequently misincorporated into DNA during replication, and they are rapidly repaired by ribonucleotide excision repair (RER). Although ribonucleotides in template DNA perturb replicative polymerases and can be considered as DNA damage, they also serve positive biological functions, including directing the orientation of mismatch repair. Here we describe a method for ribonucleotide identification by high-throughput sequencing that allows mapping of the location of ribonucleotides across the genome. When combined with specific mutations in the replicative polymerases that incorporate ribonucleotides at elevated frequencies, our ribonucleotide identification method was adapted to map polymerase usage across the genome. Polymerase usage sequencing (Pu-seq) has been used to define, in unprecedented detail, replication dynamics in yeasts. Although other methods that examine replication dynamics provide direct measures of replication timing and indirect estimates of origin efficiency, Pu-seq directly ascertains origin efficiency. The Pu-seq protocol can be completed in 12–14 d.
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Acknowledgements
A.M.C. acknowledges UK Medical Research Council (MRC) grant no. G1100074 and European Research Council (ERC) grant no. 268788-SMI-DDR.
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A.K., Y.D., K.P. and I.M. performed the experiments. A.K. and Y.D. designed the protocol and analytical methods. A.K., Y.D., A.M.C. and K.P. wrote the manuscript.
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Keszthelyi, A., Daigaku, Y., Ptasińska, K. et al. Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq). Nat Protoc 10, 1786–1801 (2015). https://doi.org/10.1038/nprot.2015.116
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DOI: https://doi.org/10.1038/nprot.2015.116
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