Action of DNA Repair Endonuclease ERCC1/XPF in Living Cells

To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary ce...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 1999-05, Vol.284 (5416), p.958-961
Main Authors: Houtsmuller, Adriaan B., Rademakers, Suzanne, Nigg, Alex L., Hoogstraten, Deborah, Jan H. J. Hoeijmakers, Vermeulen, Wim
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Bleaching
Cell Line, Transformed
Cell lines
Cell Nucleus - metabolism
CHO Cells
Cricetinae
Diffusion
DNA Damage
DNA Repair
DNA-Binding Proteins - metabolism
Endonucleases - metabolism
Fibroblasts
Fluorescence
Fundamental and applied biological sciences. Psychology
Green Fluorescent Proteins
HeLa Cells
Humans
Irradiation
Luminescent Proteins
Microscopy, Confocal
Microscopy, Fluorescence
Molecular and cellular biology
Molecular genetics
Molecules
Mutagenesis. Repair
Nucleotides
Proteins - metabolism
Recombinant Fusion Proteins - metabolism
Transfection
Ultraviolet Rays
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Summary:To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus, with a diffusion coefficient (15 ± 5 square micrometers per second) consistent with its molecular size. Ultraviolet light-induced DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.284.5416.958