Kuraoka Isao, Tanaka Kiyoji
Human Cell Biology Group, Laboratories for Organismal Biosystems, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
Methods Enzymol. 2006;408:214-23. doi: 10.1016/S0076-6879(06)08013-X.
A DNA molecule is vulnerable to many types of DNA-damaging agents of endogenous and environmental origins. Although damage to DNA can interfere not only with replication but also transcription, the majority of DNA repair and mutagenesis studies are based on the actions of DNA polymerases in DNA replication. To investigate the actions of RNA polymerase II (RNAPII) encountering a single DNA lesion on transcription elongation, we employ a transcription elongation assay using purified RNAPII and oligo(dC)-tailed templates containing a DNA lesion at a specific site. This chapter describes an analysis of whether elongating RNAPII stalls at a DNA lesion or whether RNAPII generates mutations in RNA transcripts.
DNA分子易受多种内源性和环境来源的DNA损伤剂的影响。虽然DNA损伤不仅会干扰复制，还会干扰转录，但大多数DNA修复和诱变研究都是基于DNA聚合酶在DNA复制中的作用。为了研究RNA聚合酶II（RNAPII）在转录延伸过程中遇到单个DNA损伤时的作用，我们采用了一种转录延伸测定法，该方法使用纯化的RNAPII和在特定位点含有DNA损伤的寡聚（dC）尾模板。本章描述了对延伸中的RNAPII是否会在DNA损伤处停滞，或者RNAPII是否会在RNA转录本中产生突变的分析。
