53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility - PubMed (original) (raw)

. 2008 Nov 27;456(7221):524-8.

doi: 10.1038/nature07433. Epub 2008 Oct 19.

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53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility

Nadya Dimitrova et al. Nature. 2008.

Abstract

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2(fl/-)) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.

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Figures

Figure 1

Figure 1. Requirement for 53BP1 in NHEJ of dysfunctional telomeres, but not DNA damage signaling

a, (Top) Metaphase chromosomes after deletion of TRF2 from the indicated cells. Telomeric FISH, green; DAPI, red. (Bottom) Summary of the effect of 53BP1 on telomere fusions at 120 hours after TRF2 deletion. b, In-gel assay for the 3’ overhang (left) and total telomeric DNA (right) after deletion of TRF2 from 53BP1-proficient and -deficient cells. c, Quantification of overhang signals in (b) (mean±SD; n=3). d, IF for presence of γ-H2AX at telomeres after deletion of TRF2 from 53BP1-proficent and -deficient cells. e, Immunoblots for phosphorylated ATM and Chk2 after TRF2 deletion. f, Proliferation of MEFs of the indicated genotype and treatment (mean±SD; n=3).

Figure 2

Figure 2. Optimal NHEJ of dysfunctional telomeres requires interaction of 53BP1 with H4-K20diMe

a, Schematic of the domain structure of 53BP1. b, Immunoblot for 53BP1 expression in TRF2F/−53BP1−/− cells complemented with empty vector, wild type (WT), or the Tudor mutant 53BP1-D1521A. c, Telomere fusions in metaphase spreads of TRF2F/−53BP1−/− MEFs complemented as indicated and treated with Cre as specified. d, Quantification of telomere fusions in (c) (mean±SD; n=3).

Figure 3

Figure 3. 53BP1 promotes mobility of dysfunctional telomeres

a, Schematic of the live-cell imaging experiments. b, Immunoblotting for EGFP-TRF1, mCherry-BP1-2, and TRF2. c, Co-localization of EGFP-TRF1 and mCherry-BP1-2 fluorescence signals. d, Representative traces of telomeres in indicated MEFs expressing EGFP-TRF1 and mCherry-BP1-2. Snapshots taken at indicated timepoints. e, 10 representative telomeres from indicated MEFs, treated as specified, were tracked as in (d); cumulative distance at each time-point was plotted against time. f, Frequency distribution of the cumulative distance traveled in one representative experiment in MEFs of the indicated genotype and treatment (median±SD.; n=55; p calculated using a two-tailed Mann-Whitney test). g, Frequency distribution of the maximum displacement from the starting point registered by individual telomeres during the imaging session described in (f). (median±SD and p-values calculated as in (f)). h, i, Analysis as in (f) in MEFs of the indicated genotypes and treatments, expressing EGFP-TRF1 marker only (n=50).

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