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DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei

Paola Grandi^1,*,, Michail Eltsov^2,3,*,, Inga Nielsen¹ and Ivan Raka^2,3,¶

¹ Department of Biochemistry and Molecular Biology, University of Geneva, CH1211-Geneva 4, Switzerland
² Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Albertov 4, CZ-128 00 Prague 2, Czech Republic
³ Laboratory of Gene Expression, 1st Faculty of Medicine, Charles University, Albertov 4, CZ-128 00 Prague 2, Czech Republic
^* These authors contributed equally to this study
Present address: CellZome, Meyerhofstrasse 1, 69117, Heidelberg, Germany
On leave from: Institute of Cytology, Academy of Sciences of the Russian Federation, St Petersburg, Russian Federation

View larger version (23K): [in a new window]   Fig. 1. RP-A foci form on restriction-enzyme-treated sperm pronuclei. Immunofluorescence with affinity-purified anti-RP-A70 antibodies on intact and restriction-enzyme-digested sperm pronuclei. Demembranated Xenopus sperm heads were pre-incubated for 20 minutes, either with a buffer (no addition) or with 0.05 units of each of the restriction enzymes indicated (EcoRI, DraI, PstI) and then Xenopus egg HSSs were added for 1 hour at 23°C. (B) Immunofluorescence with affinity-purified anti-RP-A70 antibodies on sperm heads preincubated with different amounts of EcoRI (0.01 units, 0.05 units, 1 unit) or with 1 unit of the rare cutting endonuclease Not1 prior to addition of HSS extracts as in (A). (C) Immunofluorescence with purified anti-RP-A70 antibodies on sperm heads pre-incubated with 0.02 units of EcoRI and then mixed with HSS and put at 4°C for 1 hour (4°C), mixed with HSS containing 5 mM AMP-PNP and incubated at 23°C for 1 hour (AMP-PNP), incubated for 1 hour with HSS at 23°C, then 40 µg ml-1 ds or ssDNA was added and incubation prolonged for an additional 30 minutes (+dsDNA, +ssDNA). The control figure (no addition) is also documented. Cy3-conjugated donkey anti-rabbit was used as a secondary antibody. (A-C) represent each a cumulative image of 10-12 confocal sections. Bars, 10 µm.

View larger version (17K): [in a new window]   Fig. 2. RP-A foci on restriction-endonuclease-treated sperm chromatin form with different types of egg extracts. Immunofluorescence with purified rabbit anti-RP-A70 antibodies on intact sperm heads (Sperm) or sperm heads pre-treated with 0.05 units of EcoRI (Sperm+RE) incubated with interphase HSS, interphase LSS or mitotic HSS for 1 hour at 23°C. A cumulative image of 10-12 confocal sections is shown for each sample. Bars, 5 µm.

View larger version (25K): [in a new window]   Fig. 3. Unscheduled DNA synthesis (UDS) occurs at RP-A foci induced by restriction endonuclease. (A) Double immunofluorescence with affinity-purified rabbit anti-RP-A70 antibodies (RPA) and with streptavidin conjugated to FITC in order to reveal incorporation of biotinylated dNTPs (dNTPs). Demembranated sperm heads (4000 heads) pretreated with 0.02 units of EcoRI (RE+HSS) or mock (HSS) are incubated for 1 hour at 23°C with HSS containing 20 µM biotinylated dATP and dUTP. Cumulative images representing 10-12 confocal sections for each fluorescence channel are shown. Most fluorescence foci owing to dNTP and RP-A signals co-localize as shown in the merged figure. It should be mentioned that, in some foci, there is an imbalance between the two color intensities, one being much higher than the other. Therefore, the yellow color in the merged figure is not that apparent in some co-localized foci (arrows). With mock pretreated sperm, the signal of the biotinylated dNTPs is barely visible. Bars, 5 µm. (B) Cesium chloride centrifugation gradient. Intact sperm heads were incubated with LSS extracts for 2 hours in the presence of [32P]dATP and with BrdCTP (LSS-HL) or without BrdCTP (LSS-LL). EcoRI-pretreated sperm heads (0.02 U EcoRI per 4000 sperm heads) were incubated with HSS extracts for 2 hours in the presence of 32P-dATP and BrdCTP (HSS RE). Equivalent amounts in sperm heads of LSS-HL and LSS-LL samples and five equivalents of HSS 43 fractions were taken from each gradient and their refractometric index as well as the total radioactivity measured. On the graph, the radioactivity expressed in arbitrary units (AU) is plotted for each fraction. The density of the fraction with the highest radioactivity value is written for each sample above the respective peaks.

View larger version (49K): [in a new window]   Fig. 4. The Xenopus homolog of Ku86 localizes in foci on restriction-enzyme-treated sperm pronuclei that largely coincide with RP-A foci and with the sites of UDS. (A) Immunofluorescence with purified anti-XKu86 antibodies on untreated (HSS) and restriction-enzyme-treated (HSS+RE) sperm heads incubated in HSS. The restriction enzyme treatment induced an accumulation of XKu86 in multiple bright foci. Double immunofluorescence with affinity-purified rabbit anti-Xenopus Ku86 antibodies (Anti-XKu86) and chicken anti-RP-A70 (RP-A) antibodies on sperm heads (4000 heads) pre-treated with 0.05 units of EcoRI (Sperm+RE) and incubated with HSS for 1 hour at 23°C. Most fluorescence RP-A and Ku foci co-localize. Owing to an imbalance between the two color intensities, the yellow color in the merged figure is not that apparent in some co-localized foci (arrows) (the same argument applies to C). (C) Double immunofluorescence with affinity-purified rabbit anti-XKu86 antibodies (XKu86) and with streptavidin conjugated to FITC to visualize the incorporation of biotinylated dNTPs (dNTPs). EcoRI-pretreated sperm were incubated with HSS in the presence of 20 µM biotinylated dATP and dUTP for 1 hour at 23°C. In the merged figure, most fluorescence foci co-localize. Cumulative images representing 10-12 confocal sections for each fluorescence channel are shown. Bars, 3 µm.

View larger version (24K): [in a new window]   Fig. 5. Depletion of RP-A, but not of XKu86, inhibits the incorporation of dNTPs in foci of EcoRI-preatreated sperm pronuclei. Double immunofluorescence with affinity-purified rabbit anti-RP-A70 antibodies (RP-A) or anti-XKu86 antibodies (XKu86) and with streptavidin conjugated to FITC to visualize the incorporation of dNTPs (dNTPs). Sperm heads (4000 heads) pretreated with 0.02 units of EcoRI were incubated with HSS that was immunodepleted with rabbit anti-RP-A antibodies in the presence of biotinylated dATP and dUTP. With RP-A-depleted HSS, no fluorescence signal of dNTPs is produced. (B) As in (A), but the HSS was immunodepleted with anti-XKu86 antibodies. Depletion of XKu86 from the HSS does not inhibit either RP-A or dNTP accumulation in the foci of the digested nuclei. In the merged figure, most fluorescence RP-A and dNTPs foci co-localize. The yellow color is not that apparent in some co-localized foci (arrows) owing to an imbalance between the two color intensities. Cumulative images of 10-12 confocal sections for each fluorescence channel are shown. Bars, 3 µm.

View larger version (127K): [in a new window]   Fig. 6. ImmunoEM analysis of RP-A foci on restriction-enzyme-treated sperm nuclei. EM micrographs of Epon (A), Lowicryl (B,D,E) and cryo (C) thin sections of EcoRI restriction enzyme-treated Xenopus sperm heads after incubation with HSS. (A) Multiple RP-A/Ku bodies (some bodies are indicated by arrows) in a sample fixed with glutaraldehyde and postfixed with osmium tetroxide. Note the structural distinction between the chromatin and RP-A/Ku bodies. (B) RP-A/Ku bodies (arrows) labeled with purified anti-RP-A70 antibodies followed by 12 nm gold-coupled anti-rabbit antibody. (C) 30 nm chromatin fibrils (arrows) seen around the RP-A/Ku body (r) (RP-A 70 labeled with 12 nm gold adduct). (D) RP-A/Ku body after DNA labeling by TdT reaction (6 nm gold) and RP-A 34 immunolabeling (12 nm gold; two such gold particles are indicated by arrows). (E) RP-A/Ku bodies (arrows) labeled for XKu86 protein (5 nm gold particles). Bar in (A), 1 µm; bars in (B-E), 100 nm.

View larger version (82K): [in a new window]   Fig. 7. RP-A and XKu86 foci form on linear duplex DNA linked to magnetic beads after incubation with HSS. Immunofluorescence with affinity purified anti-RP-A70 (RP-A) and anti-XKu86 antibodies (XKu86) on DNA bound to magnetic beads and incubated with HSS (in DNA amounts equivalent to 5000 sperm heads) for 1 hour at 23°C. The beads were extensively washed and fixed with 2% paraformaldehyde. The DNA bound to the beads was stained with DAPI. Both RP-A and XKu86 form foci all around the beads, whereas the DNA staining appears as a smoother rim. A control immunocytochemical experiment (IC) of naked beads incubated just with the extract and labeled with anti-RP-A70 antibody is shown together with the phase contrast (PC) image. An identical control image was obtained with anti-XKu86 antibodies. Bars, 5 µm.

View larger version (58K): [in a new window]   Fig. 8. RP-A and XKu are preferentially associated with the DNA sequences proximal to the ends of linear DNA linked to the beads. Western blot analysis with rabbit serum against XKu86 and rabbit anti-RP-A serum, which recognizes all three RP-A subunits (RP-A70, RP-A34 and RP-A14). The slower migrating band might correspond to the phosphorylated form of RP-A34 (RP-A34*). DNA linked to magnetic beads was incubated with HSS for 1 hour at 23°C, extensively washed and then incubated consecutively with the appropriate restriction endonuclease in order to cut off specific DNA fragments. The beads were removed with the aid of the magnet and the excised DNA fragments with the associated proteins were recovered in the supernatant, run on a 7-15% SDS-PAGE gradient gel and transferred onto a nitrocellulose membrane. Lane 1, HSS; lane 2, total proteins bound to the beads; lane 3, wash with restriction enzyme buffer; lane 4, proteins bound to end-proximal 30 bp DNA fragment; lane 6, proteins bound to end-proximal 230 bp fragment; lane 8, proteins bound to 700 bp internal DNA fragment. Lanes 5, 7 and 9, proteins remaining associated with the DNA beads after a consecutive removal of the 30 bp (lane 5), the 230 bp (lane 7) and the 700 bp fragments (lane 9). Lanes 2-9 were loaded with equivalent volumes of sample. Owing to the digestions, there is necessarily less material bound to the beads in lane 7 than in lane 5 and less material in lane 9 than in lane 7.

View larger version (25K): [in a new window]   Fig. 9. XKu86 and RP-A are required for ligation of dsDNA fragments on DNA beads incubated with HSS. Analysis of duplex DNA ligation by agarose gel electrophoresis and autoradiography. DNA bound to magnetic beads was incubated with 32P-end labeled pUC19 in the presence of mock depleted, RP-A-depleted, XKu86-depleted HSS extracts or with a buffer for 1 hour at 23°C. After incubation, the DNA beads were extensively washed with increasing concentrations of salt (up to 2 M NaCl) and detergent (up to 1% SDS). DNA beads were then equilibrated with BglII restriction enzyme buffer and digested with BglII (5 units per 100 ng DNA) for 2 hours at 37°C. The DNA fragments released from the beads were run on a 0.8% agarose gel. (A) Autoradiography: the labeled DNA fragments released by BglII digest and the linearized pUC19 are indicated on the side. (B) Ethidium bromide staining of the gel. Lane 1, mock depleted extracts; lane 2, RP-A-depleted extracts; lane 3, XKu86-depleted extracts; lane 4, buffer. Molecular weight standards are indicated on the left-hand side. The depletion of XKu86 and, to a lesser extent, RP-A from the extract inhibits end to end ligation of linear DNA fragments (arrows indicate the 7.7 kbp DNA fragments, arrowheads indicate the 5.7 kbp fragment). The asterisks indicate the presence of a contaminating DNA band migrating at 6.8 kbp. Ethidium bromide staining of the gel served as a control measure of the loading. In lane 2 in (B), the presence of the band migrating more rapidly than the 3 kbp fragment might be due to the star activity of BglII.