Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions.
Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.
Rademakers
Suzanne
S
Center for Biomedical Genetics, Medical Genetic Center-Department of Cell Biology and Genetics, Josephine Nefkens Institute, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands.
Volker
Marcel
M
Hoogstraten
Deborah
D
Nigg
Alex L
AL
Moné
Martijn J
MJ
Van Zeeland
Albert A
AA
Hoeijmakers
Jan H J
JH
Houtsmuller
Adriaan B
AB
Vermeulen
Wim
W
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D010455:Peptides; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D049452:Green Fluorescent Proteins