JCS02336 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Construction of mice with non-functional truncated 53BP1. Schematic structure of the last 12 coding exons, shown as black boxes, of the 53BP1 locus. EcoRI sites are marked ‘E’. The last 10 coding exons were replaced by a hypoxanthine phosphoribosyl transferase (HPRT) gene of the targeting vector, and a thymidine kinase (TK) gene was used for negative selection. Open boxes represent the 5¢ and 3¢ homology regions cloned in the targeting vector. An EcoRI fragment, specifically detected by a 5¢ probe external to the 5¢ homology region, is reduced to 6.5 kb after homologous recombination of the targeting vector, whereas the fragment is 9.2 kb in the wild-type (WT) locus. (B) Southern blot analysis of genomic DNA derived from the tails of the offspring of the 53BP1^+/– intercross using the 5¢ probe. Several animals only exhibit the fragment size specific for the targeted locus (6.5 kb) showing that they are 53BP1^–/– homozygous. (C) Preimmune serum control for anti-N-terminus m53BP1 staining of MEF cells. (D) Multiple sequence alignment of Tudor domains by Clustal X. TUD_DROME (the Tudor domain of Drosophila melanogaster Tudor protein), SMN1_HUMAN, MSH6_ARATH (MSH6 protein of Arabidopsis thaliana) and RBB1_HUMAN (RB binding protein 1 of human). (E) The region conserved between human and mouse 53BP1 but not-conserved in Xenopus 53BP1 is indicated. (F) Western blot analysis of HeLa cells, expressing the GFP-fusion proteins indicated, using anti-GFP antibodies. Positions of the fusion proteins are indicated by arrowheads.

• Movie 1 -

  Movie 1. Timelapse recording of micro-irradiation of a MEF nucleus containing GFP-m53BP1. Each image is a single plane; images were taken at intervals of 30 seconds.

• Movie 2 -

  Movie 2. Timelapse recording of FRAP of GFP-m53BP1 at an IRIF of a HeLa cell at 1 hour after IR. Each image is a maximum-intensity projection of 7 z-planes 1 mm apart. Images were taken at intervals of 60 seconds.