PMC

Oligomerization of purified human 53BP1 polypeptides in vitro. His-tagged polypeptides containing the indicated residues of human 53BP1 were purified to homogeneity and assayed for oligomerization by size exclusion chromatography. The calculated molecular weight (calc. MW) of each polypeptide (in thousands) is shown to the right, and the elution of molecular weight standards is shown at the bottom. The elution profile of polypeptide 1231-1606 Δ1278-1475 is consistent with an oligomeric form that dissociates into monomers during chromatography.

Heterologous oligomerization domains can restore the focus-forming activity of GFP-53BP1 fusion proteins lacking the endogenous 53BP1 oligomerization domain. (A) Diagram of the GFP-53BP1 fusion proteins containing a heterologous oligomerization domain between the GFP and 53BP1 sequences and, optionally, also an NLS. LZ, leucine zipper; TZp, modified tetrameric leucine zipper with parallel α-helices; TZa, tetramerization domain generated by fusing part of the p53 tetramerization domain to a leucine zipper, resulting in two coiled coils packing antiparallel to each other. (B) Ribbon representations of the three-dimensional structures of segments of the 53BP1 fusion proteins corresponding to the heterologous oligomerization domain and the tandem tudor domain of 53BP1. The images were generated using the programs MOLSCRIPT and RASTER3D from Protein Data Bank files: 2ZTA, GCN4 leucine zipper (LZ); 1GCL, modified tetrameric GCN4 leucine zipper with parallel α-helices (TZp); 1C26 and 2ZTA, human p53 tetramerization domain and GCN4 leucine zipper (TZa); and 1XNI, tandem tudor domain of human 53BP1 (TUDOR). (C to E) Focus-forming activities of GFP-53BP1 fusion proteins containing various segments of human 53BP1 and, optionally, a heterologous oligomerization domain and/or a D1521R substitution in the 53BP1 tudor domain, as indicated. Focus-forming activities were classified as described in the legend to Fig. .

A minimized GFP-53BP1 fusion protein forms IR-induced foci only in irradiated subnuclear compartments. (A and B) Design of the X-ray microcollimator. Silicon wafers with 1-μm-deep grooves on one of their long surfaces are stacked against each other, such that 1-μm-wide channels are formed between the plates. (A) Cross section of one wafer (top) and of several stacked wafers (bottom). (B) Position of the silicon wafers between an X-ray source and the cells to be irradiated. For clarity, only a single wafer is shown, rather than a stack of wafers. (C and D) Formation of IR-induced foci of a GFP-TZp-53BP1 fusion protein containing residues 1475 to 1635 of human 53BP1 in irradiated subnuclear compartments. The interrupted dark blue line indicates the area exposed to IR. (C) Analysis of live cells by monitoring GFP fluorescence. (D) Analysis of fixed cells by immunofluorescence for endogenous 53BP1 and for the GFP-TZp-53BP1 fusion protein. The antibody that recognizes endogenous 53BP1 does not recognize the GFP-TZp-53BP1 fusion protein. Light blue lines mark the nuclei of the cells.

Mapping of a 53BP1 region required for efficient IR-induced focus formation. (A) Diagram of the human 53BP1 protein. The boundaries of the tandem tudor and BRCT domains are indicated. The oligomerization domain (OLIG) and the region C terminal to the tudor domain (RCTD) are also indicated (see below). The polypeptide corresponding to amino acids 1220 to 1711 is recruited to sites of DNA DSBs as efficiently as full-length 53BP1. (B to E) Focus-forming activities of GFP-53BP1 fusion proteins containing the indicated residues of human 53BP1. In panels C and E, all deletions were in the context of a polypeptide fragment spanning residues 1231 to 1711 of human 53BP1. The recruitment to IR-induced foci was classified as follows: wild type (+), strong foci with almost no diffuse nucleoplasmic staining; partial (/), visible foci but also clear diffuse nucleoplasmic staining; and none (−), no visible foci. (F) Sequence conservation of residues 1251 to 1271 of human 53BP1. Above the human sequence, the residues that were substituted are indicated by a / or −, depending on whether the substitution compromised or abolished focus formation. 53BP1_hs, Homo sapiens 53BP1; Crb2_sp, S. pombe Crb2; Rad9_ce, Caenorhabditis elegans Rad9; Rad9_sc, S. cerevisiae Rad9. (G) Focus-forming activities of GFP-53BP1 fusion proteins containing residues 1231 to 1711 of human 53BP1 and the indicated amino acid substitutions.

An element corresponding to residues 1614 to 1629 of human 53BP1 is also required for efficient IR-induced focus formation. (A and B) Focus-forming activities of GFP-53BP1 fusion proteins containing various segments of human 53BP1, the modified tetrameric GCN4 leucine zipper (TZp), and, optionally, an NLS (N), as indicated. 0 Gy, nonirradiated cells. (C) Focus-forming activities of GFP-NLS-TZp-53BP1 fusion proteins containing residues 1451 to 1631 of human 53BP1 and the indicated amino acid substitutions. (D) Focus-forming activities of GFP-full-length 53BP1 fusion proteins containing the indicated amino acid substitutions. (E) Sequence conservation of residues 1591 to 1631 of human 53BP1. Above the human sequence, the residues that were substituted are indicated by a +, /, or −, depending on whether the substitution did not affect, compromised, or abolished focus formation. The C-terminal boundary of the tudor domain at residue 1602 and the boundaries of the RCTD are also shown. 53BP1 sequences are from the following species: hs, Homo sapiens; gg, Gallus gallus; xl, Xenopus laevis; tn, Tetraodon nigroviridis; dr, Danio rerio; sp, Strongylocentrotus purpuratus. (F) Sequences of the C termini of GFP-NLS-TZp-53BP1 fusion proteins with small segments of 53BP1 replaced with residues 734 to 754 of human NBS1, residues 259 to 276 of S. cerevisiae VPS27, or residues 106 to 123 of human RAP80, as indicated. The NBS1, VPS27, and RAP80 segments are in bold letters and underlined. Asterisks indicate the free C-terminal ends of the fusion proteins. (G) Focus-forming activities of the GFP-NLS-TZp-53BP1 fusion proteins containing the human NBS1, S. cerevisiae VPS27, or human RAP80 sequences. (H) The tandem tudor domain and the RCTD function as one unit. GFP-NLS-TZp-53BP1 fusion proteins containing residues 1451 to 1631 of human 53BP1 and, optionally, amino acid substitutions targeting the tudor domain (D1521R) and/or the RCTD (L1619E) were expressed in cells and scored for IR-induced focus formation, as indicated. wt, expression of a wild-type 1451-1631 human 53BP1 fragment; wt + dm, coexpression of wild-type and double mutant (D1521R and L1619E) human 53BP1 fragments; sm + sm, coexpression of single mutant D1521R and L1619E human 53BP1 fragments.