Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq)
Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq)
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DOI：
10.1038/nprot.2015.116
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发表时间：
2015-11-01
期刊：
影响因子：
14.8
通讯作者：
Carr, Antony M.
中科院分区：
文献类型：
--
作者：
Keszthelyi, Andrea;Daigaku, Yasukazu;Carr, Antony M.
核糖核苷酸在复制过程中经常错误地掺入DNANA中，并且它们通过核糖核苷酸切除修复（RERRERRER）快速修复。虽然模板DNANA中的核糖核苷酸干扰复制聚合酶，并可被认为是DNANA损伤，但它们也具有积极的生物学功能，包括指导错配修复的方向。在这里，我们描述了一种通过高通量测序进行核糖核苷酸鉴定的方法，该方法允许绘制整个基因组中核糖核苷酸的位置。当与以升高的频率掺入核糖核苷酸的复制型聚合酶中的特定突变相结合时，我们的核糖核苷酸鉴定方法适于在整个基因组中映射聚合酶的使用。聚合酶使用测序（Pu-seq）已被用于定义，在前所未有的细节，复制动态酵母。虽然其他检查复制动态的方法提供了复制时间的直接测量和起源效率的间接估计，Pu-seq直接检测起源效率。Pu-seq方案可在12-14 d内完成。
Ribonucleotides are frequently misincorporated into DNANA during replication, and they are rapidly repaired by ribonucleotide excision repair (RERRERRER). Although ribonucleotides in template DNANA perturb replicative polymerases and can be considered as DNANA 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.