hSnm1 colocalizes and physically associates with 53BP1 before and after DNA damage - PubMed (original) (raw)

hSnm1 colocalizes and physically associates with 53BP1 before and after DNA damage

Christopher T Richie et al. Mol Cell Biol. 2002 Dec.

Abstract

snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNA-damaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.

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Figures

FIG. 1.

FIG. 1.

(A) Northern blot of mRNAs from various tissues was probed with hSnm1 cDNA. A probe for β-actin was used to control for loading. (B) In vitro-translated Flag-hSnm1 protein labeled with [35S]methionine was precipitated by anti-hSnm1-protein A-agarose beads, protein A beads only, or preimmune-protein A beads. B and SN indicate beads and supernatant, respectively.

FIG. 2.

FIG. 2.

hSnm1 is localized to the nucleus. Indirect immunofluorescence of MCF-7 cells probed with hSnm1 affinity-purified polyclonal antibodies displaying (a) diffuse nuclear staining and hSnm1 bodies (as indicated by white arrow) or (b) multiple nuclear foci. Epifluorescence of MCF-7 cells expressing EGFP-hSnm1 fusion protein showing localization to (c) a nuclear body and (d) foci (transfected cell indicated by white arrow). An undamaged HT-1080 cell transiently expressing the Flag-hSnm1 fusion construct was stained with (e) anti-hSnm1 antibodies and (f) anti-Flag M2 monoclonal antibodies. (g) The two images are shown merged after DAPI staining.

FIG. 3.

FIG. 3.

Quantitative analysis of hSnm1 focus induction after DNA damage in MCF-7 cells by indirect immunofluorescence. hSnm1 antibodies were used to probe cells at various times after treatment with either ionizing radiation (IR) or 4HC. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (open bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 100 nuclei for each time point. Reported here are the averages and standard deviations of three data sets.

FIG. 4.

FIG. 4.

Quantitative analysis of hSnm1 nuclear staining during the cell cycle. (A) Histogram displaying the DNA content of asynchronous untreated MCF-7 cells analyzed by laser scanning cytometry and the observed frequencies of the different hSnm1 staining patterns corresponding to the G1, S, and G2 subpopulations. PI, propidium iodide. (B) Similar data obtained from MCF-7 cells taken 5 h after treatment with 10 Gray of ionizing radiation. The percentages of cells displaying diffuse nuclear staining with fewer than 10 foci (white bars), hSnm1 bodies (hatched bars), and more than 10 foci (black bars) were calculated after scoring at least 150 nuclei for each time point.

FIG. 5.

FIG. 5.

Colocalization of hSnm1 foci with hMre11 and BRCA1 but not hRad51, as determined by indirect immunofluorescence. HT-1080 cells were irradiated with 10 Gray and stained 2 h later with (a) anti-hSnm1 and (b) anti-hRad51; (c) merged fields after DAPI staining. HT-1080 cells were irradiated with 15 Gray and stained 9 h later with (d) anti-hSnm1 and (e) anti-hMre11; (f) merged fields after DAPI staining. Partial colocalization with BRCA1 ionizing radiation-induced foci was observed 5 h after 10 Gray, as shown by (g) anti-hSnm1 and (h) anti-BRCA1; (i) both fields merged with DAPI staining.

FIG. 6.

FIG. 6.

hSnm1 focus formation occurs normally in Nijmegen breakage syndrome cells. GM7166 primary fibroblasts were fixed 9 h after mock treatment or after exposure to 15 Gray of ionizing radiation (IR) and subjected to indirect immunofluorescence with anti-hSnm1. (a) Mock-treated cells; (b) after ionizing radiation exposure.

FIG. 7.

FIG. 7.

Colocalization of hSnm1 and 53BP1 in foci as detected by indirect immunofluorescence. MCF-7 cells were mock treated (a, b, and c) or treated with 10 Gray of ionizing radiation and fixed after 30 min (d, e, and f), after 90 min (g, h, and i), or after 5 h (j, k, and l). (a, d, g, and j) Polyclonal anti-hSnm1 staining with fluorescein isothiocyanate; (b, e, h, and k) monoclonal anti-53BP1 staining with tetramethyl rhodamine isocyanate; (c, f, i, and l) merged fields plus DAPI staining.

FIG. 8.

FIG. 8.

Colocalization of EGFP-hSnm1 protein with 53BP1. MCF-7 cells were transfected with pEGFP-hSnm1 followed by staining with anti-53BP1 and Toto-3. Localization of (a) EGFP-hSnm1 and (b) 53BP1 to a nuclear body in the same cell; (c) the two fields merged with Toto-3.

FIG. 9.

FIG. 9.

Coimmunoprecipitation of 53BP1 and hSnm1. (A) HeLa cell extracts were incubated with beads only, preimmune serum, or affinity-purified anti-hSnm1 antibodies. Polyclonal anti-53BP1 antibodies were used to detect 53BP1 by immunoblotting. (B) HEK293 nuclear extracts were prepared with or without infection with EGFP-hSnm1-expressing adenovirus and immunoblotted with monoclonal antibodies against EGFP or polyclonal antibodies against 53BP1. Immunoprecipitations (IP) were performed with these extracts with anti-hSnm1 and anti-53BP1 antibodies as shown.

FIG. 10.

FIG. 10.

Deletion analysis of hSNM1. (A) Various truncations and in-frame deletions were constructed in the hSNM1 segment of the EGFP-hSNM1 fusion gene. The solid black region indicates EGFP, and vertical lines indicate the conserved metallo-β-lactamase domain. Each construct was transfected into HT-1080 cells, and the location of the EGFP signal was determined 24 h later. (B) Expression of EGFP-hSnm1 deletion mutants in HT-1080 cells.

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