TIRR regulates 53BP1 by masking its histone methyl-lysine binding function

53BP1 is a multi-functional double-strand break (DSB) repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumors to PARP inhibitors. Central to all 53BP1 activities is its recruitment to DSBs via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, TIRR (Tudor Interacting Repair Regulator) that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, ATM phosphorylates 53BP1 and recruits RIF1 to dissociate the 53BP1–TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to DSBs. Depletion of TIRR destabilizes 53BP1 in the nuclear soluble fraction and also alters the DSB-induced protein complex centering 53BP1. These findings identify TIRR as a new factor that influences DSB repair utilizing a unique mechanism of masking the histone methyl-lysine binding function of 53BP1

Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, dis- rupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.</p

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs

The importance of phosphate ester backbone flexibility in protein-DNA recognition

The N-terminal 56 amino acid headpieces of the wild-type and mutant Y7I lac repressors and the 14-22 base pair DNAs containing the symmetric lac operator sequences can be used to study protein-DNA interaction. Phosphorus-31 NMR gives direct information as to the type of phosphate conformations present in the DNA. Thus, it can potentially provide information about protein-DNA phosphate backbone interaction. The alkaline phosphatase assay can be used to measure the dissociation constants of the headpiece:operator complexes. The wild-type operator forms the strongest complex with the wild-type headpiece while the mutant operators form weaker complexes. Relative to the wild-type headpiece, the Y7I headpiece forms weaker complexes with the operators. Although the recognition helix of this mutant headpiece is partially destroyed, its overall folding is still comparable to that of the wild-type headpiece. Most of the tertiary structure crosspeaks in the wild-type headpiece are still found in the Y7I headpiece. Results from the phosphorus-31 titrations and binding studies suggest that specific, strongly bound complexes retain the inherent conformational flexibility of the operator itself, whereas more weakly bound, but still specific, operator:protein complexes restrict the phosphate ester conformational freedom in the complex relative to the free DNA. NMR and molecular dynamics methodologies can be successfully applied to structure refinement of macromolecules. Using these methods, the 14-mer 7G 434 operator has been found to have both A- and B-like DNA conformations

Preparation of Recombinant Peptides with Site- and Degree-Specific Lysine13C-Methylation

Lysine methylation is an important post-translational modification that affects protein function; for example, the transcriptional activity of the p53 tumor suppressor protein. To facilitate structural characterization of complexes involving proteins and methylated targets by nuclear magnetic resonance spectroscopy, we devised a simple method for preparing recombinant (15)N/(13)C-enriched peptides with a (13)C-methyl-labeled methylated lysine analog. The method, which relies on the synthesis of (13)C-enriched alkylating agents, was applied to the production of 15-residue p53 peptides variously methylated at lysine analog 370. The peptides were used to probe the methylation state-dependent interactions of mono, di and trimethylated p53 with three different proteins

Solution conformation of an essential region of the p53 transactivation domain

Background:The peptide segment surrounding residues Leu22 and Trp23 of the p53 transactivation domain plays a critical role in the transactivation activity of p53. This region binds basal transcriptional components such as the TATA-box binding protein associated factors TAFII40 and TAFII60 as well as the mdm-2 and adenovirus type 5 E1B 55 kDa oncoproteins.Results:The structure of residues 14–28 of p53 was studied by nuclear magnetic resonance spectroscopy and found to prefer a two-β-turn structure stabilized by a hydrophobic cluster consisting of residues known to be important for transactivation and binding to p53-binding proteins. A peptide segment in which Leu22 and Trp23 were replaced by Gln and Ser displays a random structure.Conclusions:This structural propensity observed in the wild-type p53 peptide is important for understanding the mechanism of transcriptional activation, because very few structural data are available on transactivation domains to date. These results should aid in the design of therapeutics that could competitively inhibit binding of p53 to the oncogene product mdm-2