Modulating the body's networks could become mainstream therapy for many health issues.
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Edited by Charles M. Radding, Yale University School of Medicine, New Haven, CT, and approved January 4, 1999 (received for review October 26, 1998)
Article Figures & SI
Figures
- Figure 1
Association of ssDNA foci with recombination proteins Rad51 and RPA. (A) Visualization of ssDNA foci in human PPL fibroblasts 1 hr after 60Co irradiation with a dose of 10 Gy. Nuclear foci containing ssDNA are stained by red anti-BrdUrd antibody. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole. At this time point, no Rad51 foci are seen. (B and C) Colocalization of Rad51 protein and ssDNA foci in PPL cells at 6 hr (B) and 24 hr (C) after irradiation. Nuclei on the left show ssDNA staining (red) and the nuclei at the right anti-Rad51 immunofluorescence (green) of the same cells. (D and E) Colocalization of replication protein A detected by anti-RPA antiserum (green) and ssDNA foci (red) at 6 hr (D) and 24 hr (E) after irradiation. To facilitate the demonstration of colocalizations, the green signals were shifted purposely by one pixel to the right and by one pixel to the bottom. (F) Colocalization of Rad51/DMC1 (green) and ssDNA (red) in mouse meiotic prophase cells. (G) As a control to demonstrate the uniform labeling of meiotic DNA with BrdUrd, anti-BrdUrd staining was performed on denatured spermatogenic cells. (H) Colocalization of Rad51 (green) and ssDNA (red) on experimentally stretched chromatin fibers from a γ-irradiated culture. Because individual cells are completely destroyed during preparation, the ssDNA fibers from the optical field shown do not necessarily represent the DNA from one cell. (Bars in C and H correspond to 10 μm.)
Tables
Treatment % Nuclei with ssDNA foci Total <10 Foci >10 Foci None 1 <1 <1 None 2 1 1 None 2 1 1 Denaturation 93 11 82 Cycloheximide (10 μg/ml) (for 1 hr) 2 1 1 Actinomycin D (100 ng/ml) (for 1 hr) 3 1 2 Etoposide (0.5 μg/ml) (for 1 hr) 32 23 9 Etoposide (2 μg/ml) (for 1 hr) 43 29 14 Etoposide (6 μg/ml) (for 1 hr) 57 33 24 Etoposide (20 μg/ml) (for 1 hr) 61 15 46 MMC (0.5 μg/ml) (for 1 hr) 36 12 24 3 hr after MMC 27 11 16 6 hr after MMC 33 14 19 15 hr after MMC 29 5 24 24 hr after MMC 32 11 21 48 hr after MMC 13 1 12 1 hr after 0.5 Gy 21 16 5 1 hr after 1 Gy 36 26 10 1 hr after 2 Gy 40 20 20 1 hr after 5 Gy 45 25 20 1 hr after 10 Gy 49 15 34 6 hr after 1 Gy 22 13 9 6 hr after 5 Gy 25 14 11 24 hr after 0.5 Gy 10 3 7 24 hr after 1 Gy 14 7 7 24 hr after 2 Gy 15 6 9 24 hr after 5 Gy 21 10 11 24 hr after 10 Gy 30 16 14 -
At least 300 nuclei were analyzed for each experiment.
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Cell line treatment % Cells with No foci ssDNA foci (only) Rad51 foci (only) ssDNA and Rad51 foci* Total Co+ TGR-1 24 hr after etoposide 15 42 5 38 19 PPL 24 hr after etoposide 8 19 4 69 47 24 hr after 0.5 Gy 77 6 2 15 14 24 hr after 1 Gy 58 17 5 20 19 24 hr after 2 Gy 53 13 6 28 27 24 hr after 4 Gy 42 28 2 28 26 24 hr after 8 Gy 19 35 7 39 33 24 hr after 10 Gy 22 15 7 56 43 6 hr after 10 Gy 34 26 5 35 22 48 hr after 10 Gy 3 53 2 42 35 KRA 24 hr after 10 Gy 28 18 9 45 30 XPA None 62 19 4 15 9 -
At least 300 cells were analyzed for each experiment.
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↵* Co+ cells show >10% colocalization of Rad51 and ssDNA foci.
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Probe 1 Probe 2 PPL fibroblasts 24 hr after 10 Gy [No. (percent) of foci per nucleus stained with] XPA fibroblasts without treatment [No. (percent) of foci per nucleus stained with] 1 and 2 1 only 2 only 1 and 2 1 only 2 only Rad51 ssDNA 6.8 (35%) 5.4 (27%) 7.0 (38%) 10.7 (44%) 5.4 (22%) 8.2 (34%) RPA ssDNA 7.5 (50%) 3.7 (25%) 3.8 (25%) 9.5 (53%) 7.2 (41%) 1.1 (6%) Rad51* CREST* 0.7 (3%) 10.3 (52%) 8.7 (45%) 1.0 (4%) 10.4 (47%) 10.8 (49%) -
50 cells with at least 5 foci of each type were analyzed for each double-staining (Rad51 and ssDNA, RPA and ssDNA, Rad51 and CREST) experiment.
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↵* Because Rad51 foci and centromeres don’t interact functionally, Rad51–CREST double-staining foci are caused by chance associations.
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Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage
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