Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq.
Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq.
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DOI：
10.1038/nprot.2015.099
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发表时间：
2015-09
期刊：
影响因子：
14.8
通讯作者：
Reijns MA
中科院分区：
文献类型：
--
作者：
Ding J;Taylor MS;Jackson AP;Reijns MA
核糖核苷酸是掺入复制细胞基因组中的最常见的非规范核苷酸。它们通过核糖核酸酶（RNase）H2切割启动的核糖核苷酸切除修复有效去除。在不存在RNA酶H2的情况下，这种嵌入的核糖核苷酸可用于体内追踪DNA聚合酶活性。为了确定它们在酿酒酵母中的精确位置，我们开发了嵌入式核糖核苷酸测序（emRiboSeq），其使用重组RNA酶H2选择性地产生可连接的3 '-羟基，与利用碱性水解的替代方法相反。EmRiboSeq允许以单核苷酸分辨率对嵌入的核糖核苷酸进行可重复的、链特异性的和潜在的定量检测。该方案可以通过用特异性切口核酸内切酶替换RNase H2来适应于其他非规范碱基的全基因组定位，该方法被称为核酸内切酶测序（EndoSeq）。由于该方案需要<5天才能完成，这些方法允许DNA复制和修复的体内研究，包括复制起点和终止区的鉴定。
Ribonucleotides are the most common non-canonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by Ribonuclease (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 utilize alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. This protocol can be adapted for the genome-wide mapping of other non-canonical bases by replacing RNase H2 with specific nicking endonucleases, a method we term Endonuclease Sequencing (EndoSeq). With the protocol taking <5 days to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions.