UV-induced DNA damage results in ubiquitylation and degradation of RNA polymerase II (RNAPII). In yeast, this requires the DEF1 gene, the product of which forms a complex with the transcription-coupling repair factor, Rad26. However, whether Def1 is directly involved in RNAPII ubiquitylation has remained unclear. Here we report the establishment of a reconstituted system for studying UV-induced RNAPII ubiquitylation, which mimics the known requirements for this process in vitro. Using this system, we show that Def1 is indeed directly required for RNAPII ubiquitylation. Moreover, Def1 interacts with RNAPII in a damage-dependent manner. These results support a model in which Def1 interacts with RNAPII in response to DNA damage, recruiting the ubiquitylation machinery to enable its modification and subsequent degradation.