Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH.
The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.
Zotter
Angelika
A
Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands.
Luijsterburg
Martijn S
MS
Warmerdam
Daniël O
DO
Ibrahim
Shehu
S
Nigg
Alex
A
van Cappellen
Wiggert A
WA
Hoeijmakers
Jan H J
JH
van Driel
Roel
R
Vermeulen
Wim
W
Houtsmuller
Adriaan B
AB
D000818:Animals; D016466:CHO Cells; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000528:radiation effects; D002472:Cell Transformation, Viral; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D007700:Kinetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051758:Transcription Factor TFIIH / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D000099250:DNA Excision Repair Protein ERCC-5
D004268:DNA-Binding Proteins; D004720:Endonucleases; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007211:Indoles; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D051758:Transcription Factor TFIIH; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C007293:DAPI