UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization.
UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (gamma-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of gamma-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10-15%) up to fluences of 10 J.(-2) before saturating at higher fluences. Frequencies of gamma-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of gamma-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of gamma-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and gamma-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation.
Limoli
Charles L
CL
Department of Radiation Oncology, University of California, San Francisco, CA 94103-0806,USA. limoli@itsa.ucsf.edu
Giedzinski
Erich
E
Bonner
William M
WM
Cleaver
James E
JE
D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011995:Recombination, Genetic; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D014466:Ultraviolet Rays; D014965:X-Rays; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C094191:MRE11 protein, S cerevisiae