53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair - PubMed (original) (raw)

53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair

Francisca Lottersberger et al. Cell. 2015.

Abstract

Increased mobility of chromatin surrounding double-strand breaks (DSBs) has been noted in yeast and mammalian cells but the underlying mechanism and its contribution to DSB repair remain unclear. Here, we use a telomere-based system to track DNA damage foci with high resolution in living cells. We find that the greater mobility of damaged chromatin requires 53BP1, SUN1/2 in the linker of the nucleoskeleton, and cytoskeleton (LINC) complex and dynamic microtubules. The data further demonstrate that the excursions promote non-homologous end joining of dysfunctional telomeres and implicated Nesprin-4 and kinesins in telomere fusion. 53BP1/LINC/microtubule-dependent mobility is also evident at irradiation-induced DSBs and contributes to the mis-rejoining of drug-induced DSBs in BRCA1-deficient cells showing that DSB mobility can be detrimental in cells with numerous DSBs. In contrast, under physiological conditions where cells have only one or a few lesions, DSB mobility is proposed to prevent errors in DNA repair.

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Figures

Figure 1

Figure 1. Microtubule dynamics promote mobility of dysfunctional telomeres

(A) Schematic of the imaging approach. mCherry-BP1-2 foci at deprotected telomeres after TRF2 deletion were traced for 10 min by time-lapse microscopy. (B) Immunoblot for TRF2 and phosphorylation of Chk2 in TRF2F/F RsCre-ERT1 MEFs at 55-62 h after addition of 4-OH tamoxifen (4-OHT). (C) Images of mCherry-53BP1-2 foci with microtubule visualized with YFP-αTubulin (with γ-correction). (D) Examples of traces of mCherry-53BP1-2 foci as described in (B) and (C) and shown in Movies S1A-D. (E) and (F) Distribution of the cumulative distance travelled and MSD with SDs of all the mCherry-BP1-2 foci detected in the conditions as (C). Data obtained from three independent experiments with >10 cells/condition. Numbers below the data points are averages and SDs of the three median values from three independent experiments. Bars represent the median of all the foci (>1000) traced. P values are from two-tailed Mann-Whitney test. Symbols: (****) p<0.0001, (***) p<0.001, (**) p<0.01, (*) p<0.05, (ns) not significant. (G) Percentage of cells discarded (means and SDs from three independent experiments). P values were based on unpaired t-test. Symbols as in (F). See also Figure S1 and Table S1.

Figure 2

Figure 2. SUN1 and SUN2 promote mobility of dysfunctional telomeres

(A) Immunoblots for TRF2, SUN1, SUN2, 53BP1, and phosphorylated Chk2 in the indicated MEFs at 72 h after Hit&Run Cre. (B) TIF (Telomere dysfunction Induced Foci) assay on the MEFs described in (A). Telomeres were detected by FISH with FITC-(CCCTAA)3 probe (green). Phosphorylated H2AX (top panel), 53BP1 (middle panel), and Rif1 (bottom panel) were detected by IF (red). DAPI: DNA (blue). (C) Quantification of TIF response after Cre as assayed in (B). Cells with >10 TIFs were scored. Values are means and SDs of three independent experiments. P values were from an unpaired t-test (see legend to Figure 1). (D) Examples of traces of mCherry-53BP1-2 foci at 66-72 h after Cre (see Movies S3A-C). (E) and (F) Distribution of the cumulative distance travelled and MSDs with SDs of mCherry-BP1-2 foci in the analyzed MEFs (as (D)) in four experiments, as described in Figure 1. See also Figure S2 and Table S1.

Figure 3

Figure 3. The LINC complex promotes NHEJ of dysfunctional telomeres

(A) Metaphases showing telomere fusions in the indicated MEFs at 84 h after Hit&Run Cre. Telomeres were detected by FISH with a FITC-(CCCTAA)3 probe (green). DNA: DAPI (red). (B) Distribution of telomere fusions as in (A) at 84 and 108 h after Cre. Dots represent % fusions in individual metaphases. Bars represent the median of telomere fusions in 15 metaphases for three independent experiments (45 metaphases). P values from unpaired t-test (see legend to Figure 1). (C) In-gel assay for single-stranded telomeric DNA. Telomeric overhangs detected in situ with end-labeled 32P-(AACCCT)4 in MboI-digested genomic DNA from the indicated MEFs at 84 and 108 h after TRF2 deletion (top panel). Bottom: the DNA was denatured in situ and rehybridized with the same probe to determine the total telomere DNA. (D) Quantification of relative overhang signal as detected in (C). Values represent means for four independent experiments with SDs. The ss telomeric signal was normalized to the total telomeric DNA in the same lane. For each MEF line, the normalized no Cre value of cells was set at 100 and the post-Cre values are given relative to this value. 2way ANOVA for multiple comparisons were used to perform statistical analysis. For p value symbols see legend to Figure 1. (E) Schematic of the LINC complex and microtubules. (F) and (G) Quantification of telomere fusions in TRF2F/F MEFs treated with shRNAs to Nesprin-4 or Kif5B 96 h after Cre and analyzed as in (A) and (B). Bars represent the median % of telomeres fused in three independent experiments (20 metaphases each). (H) Quantification of telomere fusions in TRF2F/F RsCre-ERT1 and TRF2F/F Kif3AF/F RsCre-ERT1 MEFs 72 and 90 h after 4-OHT, as in (A) and (B). See also Figure S2 and Figure S3.

Figure 4

Figure 4. The mobility domain of 53BP1, but not PTIP, is required for mobility of dysfunctional telomeres

(A) Schematic of 53BP1, S/TQ site mutations, and their phenotypes. (B) Quantification of telomere fusions in the indicated MEFs complemented with the indicated 53BP1 alleles 96 h after TRF2 deletion with Hit&Run Cre (as in Figure 3). Data from >70 metaphases analyzed in four independent experiments. For each experiment, the median fusion frequency for 53BP1DB was set to 100 and all other values were normalized to this frequency. (C) MSDs with SDs of mCherry-BP1-2 foci detected in the TRF2-deleted 53BP1−/− MEFs expressing the indicated 53BP1 alleles. Data from three independent experiments. (D) Examples of traces of mCherry-53BP1-2 foci at 66-72 h after Cre in the indicated MEFs (see Movies S4A-C). (E) and (F) Distribution of the cumulative distance travelled and MSDs with SEMs of mCherry-BP1-2 foci in the indicated MEFs (as in Figure 1). Bars represent medians of the cumulative distance travelled >500 foci in the two experiments and numbers indicate the averages and SEMs of the two median values obtained in the two independent experiments. (G) Quantification of telomere fusions in the indicated MEFs at 84 and 108 h after Cre (as in Figure 3). See also Figure S4, Figure S5 and Table S1.

Figure 5

Figure 5. 53BP/LINC/microtubule promoted mobility of IR-induced DSBs

(A) Immunoblot for phosphorylation of Chk2 (as in Figure 2A) in the indicated MEFs at 1 h after 2.75 Gy IR. (B) IF for γH2AX (green) and 53BP1 (red) for cells treated as in (A). DAPI: DNA (blue). (C) Quantification of IR-induced γ-H2AX and 53BP1 foci as assayed in (B). (D) Examples of 10 min traces of mCherry-53BP1-2 foci at 1 h after IR of the cells described in (A) with or without 20 μM Taxol (see Movies S5A-D). (E-G) Percentage of cells discarded, distribution of the cumulative distance travelled, and MSDs with SDs of mCherry-BP1-2 foci detected as (D) and (E) (as in Figure 1). Data from three independent experiments. See related Figure S6 and Table S1.

Figure 6

Figure 6. SUN1/2 and dynamic microtubules promote radial formation

(A) Immunoblots for BRCA1 and γ-tubulin in the indicated MEFs (as in Figure 2A) at 144 h after infection with BRCA1 shRNA or empty vector. Olaparib was added 16 h before analysis. (B) Representative mis-rejoined chromosomes (arrowheads). DNA stained with DAPI. (C) Quantification of mis-rejoined chromosomes in the indicated MEFs (as in (A)), analyzed as in (B). Each dot represents a metaphase. Bars represent the median of mis-rejoined chromosomes in three independent experiments (10 metaphases each). P values as in Figure 3B. (D) Quantification of mis-rejoined chromosomes in the indicated MEFs 18 h with or without Taxol as in (C). (E) Quantification of mis-rejoined chromosomes in each metaphase in the indicated MEFs with or without Taxol as described in (C) and (D). All cells used in (A-F) are TRF2F/F. (F) Quantification of colony formation in the indicated cells infected with BRCA1 shRNA and treated with or without Taxol for 7 days. The curves represent the average and SEMs of two independent experiments. (G) Schematic of the role of 53BP1 in NHEJ of distant DSBs. In addition to controlling of DNA end processing, 53BP1 can affect NHEJ by increasing the mobility of DSBs. The mobility of DSBs is dependent on the LINC complex and microtubule dynamics. Dashed arrows indicate the possibility that the DDR affects the LINC complex and microtubules independent of 53BP1. See related Figure S7.

Figure 7

Figure 7. Proposed function and mechanism of 53BP1-dependent mobility of DSBs

(A) and (B) Proposed function for 53BP1-dependent mobility in promoting correct DSB repair. (A) G1: Mobility of DNA ends that have lost their association could promote their rejoining, thereby promoting NHEJ. (B) S/G2: If a DNA end loses connection with the sister chromatid and invades an ectopic locus, DSB mobility could disrupt this aberrant interaction and promote correct HDR. If the DSB is being repaired correctly using HDR on the sister chromatid, mobility will not dissociate the ends because of the presence of cohesin and basepairing. (C) Proposed models for the mechanism of 53BP1/LINC/microtubule dependent mobility of DSBs. The enlarged part of the nucleus shows 53BP1 (red) at a DSB with the ends separated. One end (top) portrays a model in which 53BP1 has a physical connection with the LINC complex (green). The LINC complex connects to dynamic microtubules and thereby moves the LINC-bound 53BP1-covered DNA end. The other end (bottom) portrays a model in which there is no physical connection between the LINC complex and 53BP1. The LINC complex associates with microtubules that ‘poke’ the nucleus. The 53BP1 associated chromatin moves more readily even when not at the periphery, perhaps because 53BP1 alters the flexibility of the chromatin fiber. See text for discussion.

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