Figure 4.
CPD repair kinetics in NER and HR pathway mutants. (a) Immunofluorescence images of germ cell nuclei from UV-C-irradiated wild-type, rad-54(ok615) and xpa-1(ok698) worms. Gonads were extruded and fixed immediately following UV-C radiation or after 14 h. Images in the upper panel represent the CPD staining (green), the nuclei DAPI staining (magenta) and an overlay of the CPD staining and DAPI in wild-type animals, resulting in a white ring where UV lesions are detected on germ cell chromatin. Although in the absence of NER in xpa-1(ok698) mutants nuclei remain white (lower panel), CPDs are removed in rad-54(ok615) mutants similar to wild type mutants (chromatin turns magenta). (b) CPD signal intensity after irradiation with 100 J/m2 UV-C was determined by fluorescence image analysis of isolated gonads. Intensity before irradiation was defined as 0 and the value immediately after UV-C as 1 for each strain, and later time points were expressed as a fraction of the initial UV-C-induced intensity. At least 10 animals were scored per data point. Error bars represent the S.E.M. Repair kinetics for CPD lesions in transition cell nuclei of wild-type hermaphrodites (t1/2 approx. 10 h) are in good agreement with repair kinetics reported for other systems. xpa-1(ok698) mutants lacking a functional NER show no significant repair, whereas rad-54(ok615) mutants exhibit a response similar to wild type mutants. (c) RAD-54::YFP foci develop slowly and persist for long in UV-C-irradiated wild-type animals. Mid–late pachytene germ cell nuclei from staged young adult wild-type animals were scored for the presence of RAD-54::YFP foci, at 5, 10, 20 min, 1, 2, 4, 8 and 16 h post 100 J/m2 of UV-C treatment