Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.
Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.
Park
M S
MS
Life Sciences Division, Los Alamos National Laboratory, NM 87545, USA. park@telomere.lanl.gov
Knauf
J A
JA
Pendergrass
S H
SH
Coulon
C H
CH
Strniste
G F
GF
Marrone
B L
BL
MacInnes
M A
MA
D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells / Q000528:radiation effects; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011993:Recombinant Fusion Proteins; D013997:Time Factors; D014157:Transcription Factors; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism*; D000099250:DNA Excision Repair Protein ERCC-5
D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5