Alternative recombination pathways in UV-irradiated XP variant cells.
XP variant (XP-V) cells lack the damage-specific polymerase eta and exhibit prolonged replication arrest after UV irradiation due to impaired bypass of UV photoproducts. To analyse the outcome of the arrested replication forks, homologous recombination (HR, Rad51 events) and fork breakage (Rad50 events) were assayed by immunofluorescent detection of foci-positive cells. Within 1 h of irradiation, XP-V cells showed more Rad51-positive cells than normal cells, while neither cell type showed an increase in Rad50 foci. Beyond 1 h, the frequency of Rad51-positive cells reached similar levels in both cell types, then declined at higher UV doses. At these later times, Rad50-positive cells increased with dose and to a greater extent in XP-V cells. Few cells were simultaneously positive for both sets of foci, suggesting a mutually exclusive recruitment of recombination proteins, or that these pathways operate at different stages during S phase. Analysis of cells containing a vector of tandemly arranged enhanced green fluorescent protein genes also showed that UV-induced HR was higher in XP-V cells. These results suggest that cells make an early commitment to HR, and that at later times a subset of arrested forks degrade into double-strand breaks, two alternative pathways that are greater in XP-V cells.
Limoli
Charles L
CL
1Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of California, 1855 Folsom St., MCB-200, San Francisco, CA 94103-0806, USA.
Giedzinski
E
E
Cleaver
J E
JE
D017766:Acid Anhydride Hydrolases; D002478:Cells, Cultured; D045643:DNA Repair Enzymes / Q000032:analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051135:Rad51 Recombinase; D011995:Recombination, Genetic; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes