Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification.
Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA. Here we analyze the biochemical interactions of specific domains of the Rad18 protein. We found that the Rad6/Rad18 complex forms stable dimers in vitro. Consistent with previous findings, both the Ring domain and a C-terminal region contribute to the Rad6 interaction, while the C-terminus is not required for the interaction with PCNA. Surprisingly we find that the C2HC zinc finger is important for interaction with ubiquitin, apparently analogous to the interactions of classical zinc fingers with ubiquitin such as found in the UBZ and UBM domains in Y-family polymerases. Finally we find that the SAP domain, but not the zinc finger domain, is capable of DNA binding in vitro.
Notenboom
Valerie
V
Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Hibbert
Richard G
RG
van Rossum-Fikkert
Sarah E
SE
Olsen
Jesper V
JV
Mann
Matthias
M
Sixma
Titia K
TK
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; C485953:Rad18 protein, mouse; D025801:Ubiquitin; D004247:DNA; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases