Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC.
Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A in vivo. The majority of DDB2 and CUL4A diffuse in the nucleus with a diffusion rate consistent with a high molecular mass complex. Essentially all DDB2 binds to UV-induced DNA damage, where each molecule resides for approximately 2 minutes. After the induction of DNA damage, DDB2 is proteolytically degraded with a half-life that is two orders of magnitude larger than its residence time on a DNA lesion. This indicates that binding to damaged DNA is not the primary trigger for DDB2 breakdown. The bulk of DDB2 binds to and dissociates from DNA lesions independently of damage-recognition protein XPC. Moreover, the DDB2-containing E3 ubiquitin ligase is bound to many more damaged sites than XPC, suggesting that there is little physical interaction between the two proteins. We propose a scenario in which DDB2 prepares UV-damaged chromatin for assembly of the NER complex.
Luijsterburg
Martijn S
MS
Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Nuclear Organisation Group, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.
Goedhart
Joachim
J
Moser
Jill
J
Kool
Hanneke
H
Geverts
Bart
B
Houtsmuller
Adriaan B
AB
Mullenders
Leon H F
LH
Vermeulen
Wim
W
van Driel
Roel
R
D000818:Animals; D002460:Cell Line; D044842:Cullin Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D051379:Mice; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014466:Ultraviolet Rays
C114234:CUL4A protein, human; D044842:Cullin Proteins; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; C086984:XPC protein, human; D004247:DNA; D044767:Ubiquitin-Protein Ligases