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Science 339 (6120): 700-704

Copyright © 2013 by the American Association for the Advancement of Science

53BP1 Regulates DSB Repair Using Rif1 to Control 5' End Resection

Michal Zimmermann1,2, Francisca Lottersberger1, Sara B. Buonomo3, Agnel Sfeir1,*, and Titia de Lange1,{dagger}

1 Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY 10065, USA.
2 Central European Institute of Technology and Faculty of Science, Masaryk University, Brno, Czech Republic.
3 European Molecular Biology Laboratory, Mouse Biology Unit, Monterotondo, Italy.


Figure 1
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Fig. 1. Rif1 recruitment requires the S/TQ ATM/ATR target sites of 53BP1. (A) Detection of 53BP1 and Rif1 at dysfunctional telomeres in Cre-treated SV40 large T antigen (SV40-LT) immortalized TRF2F/–53BP1–/– MEFs expressing 53BP1 mutant alleles [shown in (C)]. Indirect immunofluorescence (IF) for 53BP1 and Rif1 (red) was combined with telomeric TTAGGG fluorescence in situ hybridization (FISH) (green). Blue: 4',6-diamidino-2-phenylindole DNA stain. (B) Quantification of 53BP1 and Rif1 telomere dysfunction–induced foci (TIFs) (21) detected as in (A). Data represent means of three experiments ±SDs (≥70 cells per experiment). **, P< 0.05 (two-tailed paired Student's t test). (C) Schematic of the 53BP1 mutant alleles and the role of the N-terminal S/TQ sites in the recruitment of Rif1.

 

Figure 2
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Fig. 2. Rif1 promotes telomeric NHEJ without affecting telomere mobility. (A) Metaphase chromosomes of Cre-treated SV40-LT immortalized TRF2F/FRif1+/+ and TRF2F/FRif1F/F MEFs showing NHEJ-mediated telomere fusions detected by chromosome orientation FISH. Telomeres synthesized by leading-end DNA synthesis are in red; lagging-end telomeres are in green. (B) Quantification of telomere fusions as determined in (A) at 96 and 120 hours after Cre. Data represent means of three independent experiments ±SDs (>3000 telomeres per experiment). **, P < 0.01 based on two-tailed paired Student's t test. (C) Distributions of telomere fusions per metaphase at 96 hours after Cre for experiments shown in (B). (D) Distribution of cumulative distances traveled by mCherry-53BP11220-1711 foci in the indicated cell lines. Red lines represent medians. **, P < 0.0001 (two-tailed Mann-Whitney test).

 

Figure 3
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Fig. 3. Rif1 blocks 5' end resection at dysfunctional telomeres. (A) Telomeric overhang assays on TRF2F/FRif1+/+, TRF2F/FRif1F/F and TRF2F/–53BP1–/– MEFs. Native in-gel hybridization of MboI/AluI digested DNA with end-labeled [AACCCT]4 (top) and re-hybridization with the same probe after denaturation in situ (bottom). Dashed lines represent the bulk of free (unfused) telomeres used for quantification. (B) Quantification of overhang assays as in (A). Overhang signals in no Cre samples was set at 100%. (C and D) Overhang assays on TRF2F/FRif1F/+Lig4–/–, TRF2F/FRif1F/FLig4–/–, and TRF2F/–53BP1–/– MEFs and quantification as in (B). (E and F) Overhang assays on TRF1F/FTRF2F/FRif1+/+ and TRF1F/FTRF2F/FRif1F/F MEFs and quantification. (G and H) Overhang assays to measure dependency on CtIP, BLM, and Exo1 and quantification. Cells infected with either pMX or pSR with or without the indicated shRNAs and treated with Cre for 96 hours. Samples with empty vectors and no Cre (ref.) were used as references. Data in [(B), (D), (F), and (H)] represent means of ≥3 experiments ±SDs. **, P < 0.05 (two-tailed paired Student's t test). MEFs are SV40-LT immortalized.

 

Figure 4
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Fig. 4. Rif1 inhibits resection at DSBs and promotes radial formation. (A) IF for {gamma}-H2AX (red) and MYC-RPA32 (green) in Cre-treated SV40-LT immortalized Rif1F/F and Rif1F/F53BP1–/– cells expressing MYC-RPA32 treated with zeocin (100 μg/ml, for 1 hour; 2 hours before analysis). (B) Percentage of {gamma}-H2AX positive cells in experiments as in (A). (C) Percentage of cells [as in (A)] scored positive when containing at least five {gamma}-H2AX foci colocalizing with RPA. (D) IF for {gamma}H2AX (green) and BARD1 (red) in Rif1F/F and Rif1F/F53BP1–/– MEFs. Cells and treatment as in (A). (E) Percentage of {gamma}-H2AX-positive cells in experiments in (D). (F) Percentage of cells in (D) containing >5 BARD1/{gamma}-H2AX colocalizing foci. (G) Examples of misrejoined and radial chromosomes (arrowheads) in BRCA1sh/PARPi-treated Rif1F/F cells with or without Cre. (H) Percentages of chromosomes that are misrejoined in the indicated genotypes and treatments. Data in (B), (C), (E), (F), and (H) are means of three to five experiments ±SDs. **, P < 0.05 (two-tailed paired Student's t test).

 


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