DNA damage-induced Def1-RNA polymerase II interaction and Def1 requirement for polymerase ubiquitylation in vitro.
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.
Reid
James
J
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Svejstrup
Jesper Q
JQ
D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004249:DNA Damage; D056827:Endosomal Sorting Complexes Required for Transport; D017353:Gene Deletion; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C451742:DEF1 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D025801:Ubiquitin; C088086:rad26 protein, S pombe; D043743:Ubiquitin-Protein Ligase Complexes; D012319:RNA Polymerase II; C469287:RSP5 protein, S cerevisiae