期刊
NATURE PROTOCOLS
卷 10, 期 11, 页码 1786-1801出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.116
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资金
- UK Medical Research Council (MRC) [G1100074]
- European Research Council (ERC) [268788-SMI-DDR]
- MRC [G1100074] Funding Source: UKRI
- Medical Research Council [G1100074] Funding Source: researchfish
- Grants-in-Aid for Scientific Research [15H06032] Funding Source: KAKEN
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.
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