Assays for transcription elongation by RNA polymerase II using oligo(dC)-tailed template with single DNA damage

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.