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First published online 19 April 2005
doi: 10.1242/jcs.02336

Journal of Cell Science 118, 2043-2055 (2005)
Published by The Company of Biologists 2005
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53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin

Fiona Pryde1,*, Shirin Khalili1,*, Kathryn Robertson1, Jim Selfridge2, Ann-Marie Ritchie2, David W. Melton2, Denis Jullien1 and Yasuhisa Adachi1,{ddagger}

1 The Wellcome Trust Centre for Cell Biology, The Institute of Cell and Molecular Biology, The University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JR, UK
2 Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, The University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, UK



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  		Fig. 1. Dynamics of GFP-m53BP1 in vivo. (A) HeLa cells stably expressing GFP fused to full-length mouse 53BP1 (GFP-m53BP1) were untreated or exposed to ionising radiation (IR; 10 Gy of X rays). After recovery for 1 hour, cells were fixed and stained for phosphorylated histone H2AX ({gamma}H2AX). (B) Extracts were prepared from the HeLa cell line stably expressing GFP-m53BP1 and analysed by western blotting with anti-GFP antibodies (lanes 1, 2) and with antibodies against the C-terminus of mouse 53BP1 (lanes 3,4,5). Equal amounts of extracts from mouse NIH3T3 cells (lane 3) and un-transfected HeLa cells (lanes 1, 4) were run as controls. Samples shown here were separated on the same gel. GFP-m53BP1 migrates slightly slower than the endogenous m53BP1, which makes the band in lane 5 broader than the band in lane 4. Note that the two proteins were not separable. (C) Accumulation of GFP-m53BP1 at the nuclear region damaged by high power laser. Wild-type MEF (mouse embryonic fibroblast) cells were transfected with GFP-m53BP1 and irradiated with a laser at the region indicated in red. After GFP-m53BP1 stripe formation, the cells were fixed and stained for phosphorylated ({gamma}) H2AX with Cy3 (red). The bright spots stained with DAPI are condensed heterochromatin of mouse cells. (D) Time lapse recording of laser-induced stripe formation (the stripe is marked by an arrowhead). Wild-type MEF cells, transfected with GFP-m53BP1 or GFP-M, were micro-irradiated and maintained at 37°C. Sequential images were captured and used to create a movie series. Selected frames are shown, along with the corresponding time (in seconds) after micro-irradiation.

 


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  		Fig. 2. GFP-m53BP1 complements a defect of cells lacking 53BP1. (A,B) Homozygous mutant mice that lack 10 exons of the 53BP1 gene, encoding the last 702 amino acids, were produced (see supplementary material Fig. S1). Western blot analysis of liver, testis and brain extracts from wild-type (+/+) and the mutant (–/–) mice with affinity-purified anti-C-terminus 53BP1 (residues 1255-1957) antibodies (A) or with anti-N-terminus (residues 1-355) antibodies (B). Each well was loaded with 25 µg of the total extracts indicated. The extracts from the mutant mice lack proteins recognized by the antibodies. In A, a faint smear in the –/– brain lane (marked by x) is non-specific binding to a highly expressed protein in the brain that corresponds to the protein band (marked by x) in B, lower panel. (C) Similar analysis was performed in MEF cells. Truncated 53BP1 (Tr) is detected in the total extracts of MEF cells from the mutant mice, when anti-N-terminus serum is used. The amount of the truncated protein is significantly lower than the wild-type protein (WT). Preimmune serum for the anti-N-terminus did not react with any proteins. (D) MEF cells were exposed to 10 Gy of X rays, allowed to recover for 1 hour, fixed and subjected to staining with the antibodies indicated. In contrast to the wild-type 53BP1, which shows focal distribution, the truncated 53BP1 in mutant MEFs (–/–) does not show focal distribution when probed with anti-N antibodies. DNA was counterstained with DAPI. (E) GFP-m53BP1 fusion protein (green) was transiently expressed in the mutant MEF (–/–), irradiated with 10 Gy X rays, fixed and stained with anti-phospho-Chk2 antibodies (Chk2T68P; Cy3 in red). Bar, 10 µm.

 


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  		Fig. 3. FRAP analysis of GFP-m53BP1. (A) Fluorescence recovery kinetics of GFP-m53BP1 in non-irradiated HeLa cells. Images before bleaching (–1.5 s), immediately after the photobleach event (0 s), and later in the time course. The photobleached region is indicated by an arrowhead. (B) Relative intensities are plotted versus time in seconds (n=10). (C) FRAP analysis of GFP-NLS serves as a control (n=12). (D) Photobleaching and recovery of GFP-m53BP1 focus (indicated by an arrowhead) induced by exposure to 10 Gy of X rays. (E) Relative intensities of the GFP-m53BP1 foci plotted versus time after photobleaching (n=8). Values represent means±s.d. Bars, 10 µm.

 


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  		Fig. 4. Essential domain of 53BP1 for targeting to the nuclear foci induced by IR. (A) Schematic diagram of GFP-fusion constructs. The various portions of mouse 53BP1 were fused at their N-terminus (in some cases at their C-terminus) to GFP and tested for their ability to target (+) or not (–) to the IR-induced 53BP1 foci. The positions of the RG stretch (RG), Tudor motifs, nuclear localization signal (NLS) and BRCT motifs are boxed. (B) Amino acid sequence alignment of the RG-stretch in mouse, human and Xenopus 53BP1. (C-M) HeLa cells were transfected with the GFP-fusion constructs indicated, treated with (+) or without (–) irradiation of 10 Gy X rays and observed under a fluorescent microscope. (N) Endogenous 53BP1 was visualized with an antibody to the N-terminus of 53BP1 (red) that does not recognize the M (minimum) domain, in NIH3T3 cells expressing GFP-M (green). (O,P) GFP-M fusion protein (green) was transiently expressed in the mutant MEF (–/–), irradiated with 10 Gy X rays, fixed, and stained with anti-{gamma}H2AX antibodies (Cy3 in red) or with anti-N-terminus m53BP1 (Cy3 in red). Bars, 10 µm.

 


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  		Fig. 5. RNase treatment dissociates 53BP1 from the damage-induced foci. (A,B) NIH3T3 cells exposed to X rays were permeabilised with 0.5% Tween 20, treated with (B) or without (A) 1 mg/ml RNase A, fixed, and stained for 53BP1. The level of 53BP1 associated with the foci reduces significantly after treatment with RNase A. (C) Western blot for the samples treated with or without RNase A shows that this reduction is not due to degradation of 53BP1. Equal amounts of total protein were loaded in each lane. The blots for tubulin confirm equal loading. (D) Treatment with an endoribonuclease for RNA/DNA duplexes (RNase H) does not dissociate 53BP1 from the foci. (E,F) In contrast to 53BP1, phosphorylated {gamma}-H2AX foci are not affected by RNase treatment. (G,H) X-irradiated HeLa cells were treated either with 0.5% Tween 20 alone (G) or with 0.5% Tween 20 plus RNase A (H). In the presence of 2% Tween 20 (I,J), 53BP1 was dissociated specifically by RNase A treatment (J). (K-Y), HeLa cells were transfected with plasmids expressing the GFP-M domain fusion or GFP-{Delta}RG (M domain lacking the RG stretch). The cells were exposed to 10 Gy of X rays, incubated at 37°C for the period indicated (K-N, for 20 minutes; O-U, for 1 hour; V-Y, for 2 hours) and permeabilised with Tween 20. Cells were treated with (L,N,P,R,T,U,W,Y) or without (K,M,O,Q,V,X) RNase A after permeabilisation. (S) Cells were treated with RNase H. (T,U) After RNase A treatment, cells were treated with RNase inhibitor and then incubated with nuclear RNA (T) or with tRNA (U). Bars, 10 µm.

 


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  		Fig. 6. Association of 53BP1 with RNA. RNA was extracted from 53BP1 immunoprecipitates (lanes 2 and 4) and from normal rabbit IgG control (lanes 3 and 5), labelled with [32P]pCp, separated on a 6% denaturing polyacrylamide gel, and auto-radiographed. Total nuclear RNA was labelled and used as a control (lane1). After incubation with HeLa nuclear extracts, immuno-affinity beads were washed in two different buffers. In one set (lanes 2 and 3), 100 mM sodium acetate was used as a salt. In the other set (lanes 4 and 5), 150 mM sodium chloride was used. Several bands were enriched in the 53BP1 precipitates (marked with asterisks). Molecular weight marker (MW) was end-labelled fragments of pBR322 digested with MspI. Sizes are indicated as nucleotide length.

 

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© The Company of Biologists Ltd 2005