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. 1999 Jan 11;144(1):11-20.
doi: 10.1083/jcb.144.1.11.

Sequestration of mammalian Rad51-recombination protein into micronuclei

T Haaf  1 , E Raderschall, G Reddy, D C Ward, C M Radding, E I Golub
Affiliations

Sequestration of mammalian Rad51-recombination protein into micronuclei

T Haaf et al. J Cell Biol. .

Abstract

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after gamma irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.

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Figures

Figure 1
Figure 1
Dynamic changes in the higher-order nuclear organization of Rad51 foci after DNA damage. TGR-1 fibroblasts were irradiated with 10 Gy of 137Cs and then allowed to recover for various times. Rad51 protein is stained by green immunofluorescence, nuclei are counterstained with DAPI. 3 h after irradiation (a), Rad51 foci are distributed throughout the entire nuclear volume. After 16 h (b), clusters and linear strings of Rad51 foci are formed. After 30 h (c), Rad51 clusters move towards the nuclear periphery and are eliminated into MN. 2 d after γ irradiation (d), all Rad51 foci are excluded into MN and the nucleus is cleared of detectable Rad51-protein foci. Note the double dot appearance of Rad51 foci (a) and the paired higher-order structures (b–d). Bar, 10 μm.
Figure 2
Figure 2
Genome fragmentation in Rad51-positive MN and DNA fibers. (a and b) MN, 2 d after treatment of TGR-1 cultures with etoposide (a) and colcemid (b), contain Rad51 protein and fragmented DNA. Top nuclei, green anti-Rad51 antibody; bottom nuclei, red FISEL staining of the same cell. Probably due to the proteolytic cleavage of Rad51 during apoptosis, FISEL-positive MN display a dispersed Rad51 immunofluorescence. (c and d) DNA fibers extruded from TGR-1 nuclei, 2 d after γ irradiation. Chromatin is counterstained with DAPI. The Rad51 signals appear as beads on a string on the linear DNA molecule (c, green), whereas the remaining nuclei are devoid of detectable Rad51 staining. DNA strand breaks in chromatin fibers are detected by FISEL (d, red). Some fibers appear to condense into MN (arrow). Bar, 10 μm.
Figure 3
Figure 3
Cell cycle arrest and DNA repair synthesis of Rad51-foci–positive cells. (a) Simultaneous staining of Rad51 protein (green) and replicating DNA (red) in an exponentionally growing, untreated XPA fibroblast culture. BrdU was incorporated into DNA for 30 h and detected with red anti-BrdU antibody. Note that the Rad51-positive cell is devoid of BrdU label. (b) Colocalization of Rad51 foci (green) with DNA repair sites after DNA damage. Human PPL fibroblasts were irradiated with a dose of 1 Gy. BrdU was added 24 h postirradiation for 2 h. In contrast to replicating cells, which show strong overall BrdU staining, cells undergoing DNA repair synthesis exhibit only few discrete BrdU-incorporation sites (red). Bar, 10 μm.
Figure 4
Figure 4
Frequencies of Rad51-foci–positive nonreplicating cells (solid black bars) after γ irradiation. Human PPL fibroblasts were irradiated with doses of 1 Gy and 10 Gy and analyzed at different times postirradiation. Cells undergoing DNA replication at a specific time point were scored as BrdU+, whereas nonreplicating cells were BrdU−. Simultaneously, the cells were stained for the presence (Rad51+) or absence (Rad51−) of Rad51 foci. At least 300 cells were analyzed for each experiment.
Figure 5
Figure 5
Spatial relationships between Rad51 foci and Rad52, Gadd45, and RPA after DNA damage. TGR-1 nuclei, 3 (left), 16 (middle), and 30 h (right) after γ irradiation. Rad51-protein foci show green immunofluorescence. Rad52 (a), Gadd45 (b), and RPA (c) are detected by red antibody probes. Nuclei are counterstained with DAPI. Only RPA colocalizes with Rad51 and segregates into MN. Bar, 7 μm.
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