Sequential assembly of the nucleotide excision repair factors in vivo.
Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.
Volker
M
M
Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL, Leiden, The Netherlands.
Moné
M J
MJ
Karmakar
P
P
van Hoffen
A
A
Schul
W
W
Vermeulen
W
W
Hoeijmakers
J H
JH
van Driel
R
R
van Zeeland
A A
AA
Mullenders
L H
LH
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D005347:Fibroblasts / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008954:Models, Biological; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism*
D046911:Macromolecular Substances; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D011088:DNA Ligases