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Nat Protoc. 2015 Sep;10(9):1433-44. doi: 10.1038/nprot.2015.099. Epub 2015 Aug 27.

Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq.

Author information

1
Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK.

Abstract

Ribonucleotides are the most common noncanonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by RNase H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae, we developed embedded ribonucleotide sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3'-hydroxyl groups, in contrast to alternative methods that use alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. For the genome-wide mapping of other noncanonical bases, RNase H2 can be replaced with specific nicking endonucleases in this protocol; we term this method endonuclease sequencing (EndoSeq). With the protocol taking <5 d to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions.

PMID:
26313479
PMCID:
PMC4876909
DOI:
10.1038/nprot.2015.099
[Indexed for MEDLINE]
Free PMC Article

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