BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping

Abstract

The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals--a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction-induced foci. Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction.

Figure 1

BRCA2 is required to recruit RAD51 to the telomeres during S/G2. (a) ChIP analyses were carried out using HeLa 1.2.11 extracts prepared at 0, 2, 4, 6, 8 hours after release from double-thymidine block or untreated cells (ASY). Error bars represent SD of three independent experiments. (b) Extracts prepared from HeLa 1.2.11 cells collected six days after the first transfection with control GFP or BRCA2 siRNAs were processed for ChIP analysis.

Figure 2

Conditional deletion of Brca2 causes telomere shortening. (a) Diagrammatic representation of the Brca2⁺ and Brca2^sko alleles. Wild type and sko alleles of Brca2 were visualized on a Southern blot of mouse genomic DNA. Cleavage of the Brca2^sko allele following Cre treatment was detected by genomic PCR with the pair of primers indicated. (b) Cell extracts were prepared from TBX2-immortalized Brca2^sko/− MEFs at the indicated times after selection and analyzed by Western blotting as indicated. SMC1 was used as a loading control. (c) − (e) Q-FISH analysis of telomere length distribution in Brca2^sko/− MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses and analyzed two, six and ten days after start of selection. Average telomere length values are shown with SEM. n, number of telomeres analyzed for each sample.

Figure 3

BRCA2 and RAD51 are required for telomere length maintenance in MEFs. (a) Trp53^−/− MEFs or Terc^−/− G4 MEFs immortalized by p53 knock-down were infected with retroviruses expressing control GFP or RAD51 shRNAs, then selected with puromycin. Cell extracts prepared six days after the first infection were analyzed by Western blotting as indicated. Recombinant human RAD51-His₆ served as control. (b) Q-FISH analysis of telomere length distribution in cells treated as in (a). Average telomere length values, indicated by white bar, are shown along with SEM. Statistical analyses were performed using the Wilcoxon signed rank test. ***, P<0.0001.

Figure 4

Increased telomere fragility in BRCA2- and RAD51-deficient MEFs. (a) Representative images of fragile telomeres identified by the presence of multiple telomeric signals (MTS, yellow arrowheads) in Brca2-deleted and RAD51 shRNA-depleted MEFs. Bar, 10 μm. (b) − (d) MEFs of the indicated genotypes were treated with retroviruses encoding Cre or shRNAs in the presence (APH) or absence (DMSO) of aphidicolin. The frequency of MTS was quantified in metaphase spreads following colcemid arrest. Error bars represent SD of 3 independent experiments. n, number of chromosomes scored for each sample.

Figure 5

Homologous recombination activities and the telomeric factor TRF1 act independently in facilitating telomere replication. (a) Western blot detection of mouse TRF1 and RAD51 in LT-immortalized TRF1^F/F MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses 3 days post-selection. Tubulin was used as loading control. (b) MTS quantification in metaphase spreads following colcemid arrest cells treated as in (a). Error bars represent SD of three independent experiments. n, number of chromosomes scored for each sample. Statistical analyses were performed on total MTS using an unpaired two-tailed t-test, P<0.0001. (c) Cell proliferation assays for cells treated as in (a). Error bars represent SD of three independent experiments.

Figure 6

Telomere dysfunction-induced foci (TIFs) accumulate in BRCA2- and RAD51-deficient MEFs and in Brca2-deficient mammary tumors. (a) Immunofluorescence detection of γH2AX (green) combined with FISH staining of the telomeres (red) in Brca2^sko/− MEFs immortalized by TBX2 overexpression and treated with either empty vector (− Cre) or Cre recombinase (+ Cre). Enlarged image depicts the area marked with yellow rectangle. TIFs, the sites of γH2AX co-localization with telomeres, are indicated by yellow arrowheads. Bar, 10 μm. (b) The percentage of metaphase nuclei exhibiting <3 or ≥3 TIFs was determined for at least 50 metaphases prepared as in (a) and collected 6 days post-selection. Error bars represent SD of two independent experiments. Statistical analyses were performed using an unpaired two-tailed t-test. **, P<0.001. (c) As (b), but TIFs were analyzed in Trp53^−/− MEFs treated with control GFP shRNA and an shRNA against mouse RAD51. Error bars represent SD of two independent experiments. Statistical analyses were performed using an unpaired two-tailed t-test. *, P<0.05. (d) Immunofluorescence detection of 53BP1 (green) combined with telomeric FISH staining (red) in sections from a K14Cre-Trp53^F/FBrca2^F/F mammary tumor. Bar, 10 μm. (e) Enlarged images of TIFs, marking sites of 53BP1 co-localization with telomeres, or non-TIFs from mouse mammary tumors. Bar, 3 μm. (f) The percentage of cells containing one or more 53BP1 foci that co-localized with telomeres into TIFs was determined relative to the total number of cells analyzed (% of TIF positive cells) using paraffin-embedded sections from mouse K14Cre-Trp53^F/F, K14Cre-Trp53^F/FBrca1^F/F or K14Cre-Trp53^F/FBrca2^F/F tumors. At least 100 cells were scored for each tumor. Error bars represent SD. P values were obtained using an unpaired two-tailed t-test. ***, P<0.0001.

Figure 7

BRCA2 function, but not that of BRCA1, is essential for telomere maintenance in human breast tumors. (a) Conditional deletion of Brca2, but not Brca1 triggers telomere shortening in MEFs. Q-FISH analysis of telomere length distribution in Brca1^SCo/− and Brca2^sko/− MEFs treated with Cre (+ Cre) and control (− Cre) retroviruses and analyzed six days after start of selection. Average telomere length values, indicated by white bar, are shown along with SEM. Statistical analyses were performed using the Wilcoxon signed rank test. NS, P>0.05; ***, P<0.0001. (b) Quantification of MTS frequency in metaphase spreads from cells treated as in (a). Error bars represent SD from 2 independent experiments. n, number of chromosomes scored for each sample. (c) Q-FISH analysis of a breast tumor microarray collection consisting of 12 BRCA1-null and 10 BRCA2-null human breast tumors. The intensity of FISH telomeric signal was quantified for at least 6,000 cells for each type of tumor. a.u., arbitrary fluorescence units. Average telomere intensity values, indicated by white bar, are shown along with SEM. Statistical analyses were performed using the Wilcoxon signed rank test. ***, P<0.0001. (d) Quantification of the relative changes in the frequency of cells with short telomeres (< 20 a.u.) in BRCA1- and BRCA2-mutated human breast tumors.