title: Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq.
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.
authors: Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK.
authors: James
authors: http://orcid.org/0000-0001-7273-9646
authors: J
authors: Ding
authors: Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK.
authors: Martin S
authors: MS
authors: Taylor
authors: Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK.
authors: Andrew P
authors: AP
authors: Jackson
authors: Medical Research Council (MRC) Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh, UK.
authors: Martin A M
authors: http://orcid.org/0000-0002-5048-2752
authors: MA
authors: Reijns
mesh_terms: D002874:Chromosome Mapping; D023281:Genomics / Q000379:methods*; D016914:Ribonuclease H / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D017421:Sequence Analysis / Q000379:methods*
chemical_list: C091139:ribonuclease HII; D016914:Ribonuclease H