The RNF168 paralog RNF169 defines a new class of ubiquitylated histone reader involved in the response to DNA damage.

Site-specific histone ubiquitylation plays a central role in orchestrating the response to DNA double-strand breaks (DSBs). DSBs elicit a cascade of events controlled by the ubiquitin ligase RNF168, which promotes the accumulation of repair factors such as 53BP1 and BRCA1 on the chromatin flanking the break site. RNF168 also promotes its own accumulation, and that of its paralog RNF169, but how they recognize ubiquitylated chromatin is unknown. Using methyl-TROSY solution NMR spectroscopy and molecular dynamics simulations, we present an atomic resolution model of human RNF169 binding to a ubiquitylated nucleosome, and validate it by electron cryomicroscopy. We establish that RNF169 binds to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs that interact with the nucleosome acidic patch. This three-pronged interaction mechanism is distinct from that by which 53BP1 binds to ubiquitylated H2A-Lys15 highlighting the diversity in site-specific recognition of ubiquitylated nucleosomes

The MMS22L-TONSL Complex Mediates Recovery from Replication Stress and Homologous Recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks

A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair

Abstract The Mre11-Rad50 protein complex is an initial responder to sites of DNA double strand breaks. Many studies have shown that ATP binding to Rad50 causes global changes to the Mre11-Rad50 structure, which are important for DNA repair functions. Here we used methyl-based NMR spectroscopy on a series of mutants to describe a dynamic allosteric pathway within Rad50. Mutations result in changes in the side chain methyl group chemical environment that are correlated with altered nanosecond timescale dynamics. We also observe striking relationships between the magnitude of chemical shift perturbations and Rad50 and Mre11 activities. Together, these data suggest an equilibrium between a ground state and an “active” dimerization competent state of Rad50 that has locally altered structure and dynamics and is poised for ATP-induced dimerization and eventual ATP hydrolysis. Thus, this sparsely populated intermediate is critical for Mre11-Rad50-directed DNA double strand break repair

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed

Nucleosome Acidic Patch Promotes RNF168- and RING1B/BMI1-Dependent H2AX and H2A Ubiquitination and DNA Damage Signaling

Justin W. Leung, Poonam Agarwal, Fade Gong, Aaron D. Robison, Ilya J. Finkelstein, Kyle M. Miller, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, United States of AmericaJustin W. Leung, Poonam Agarwal, Fade Gong, Aaron D. Robison, Ilya J. Finkelstein, Kyle M. Miller, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of AmericaMarella D. Canny, Daniel Durocher, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, CanadaHistone ubiquitinations are critical for the activation of the DNA damage response (DDR). In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub). The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.The IJF laboratory was supported by the Welch Foundation (F-1808) and the Cancer Prevention Research Institute of Texas (CPRIT). DD is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 1) in the Molecular Mechanisms of Genome Integrity. The DD laboratory was supported by CIHR grant MOP89754. The research in the KMM laboratory was supported in part by start-up funds from the University of Texas at Austin and from CPRIT (R116). KMM is a CPRIT scholar. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Molecular BiosciencesInstitute for Cellular and Molecular BiologyEmail: [email protected]

The nucleosome acidic patch and histone ubiquitination.

<p>Summary of our results within the context of the nucleosome structure. The acidic patch is required for RNF168- and RING1B/BMI1-dependent histone ubiquitination and LANA inhibits these processes. See text for details. Nucleosome structure was created in Pymol as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004178#pgen.1004178-Chen1" target="_blank">[20]</a>.</p

Mutation of the acidic patch impairs human H2AX and H2A ubiquitination.

<p>(<b>A</b>) Schematic of all H2AX lysines (K) and mutant derivatives. allR represents an all lysine (K) to arginine (R) version of H2AX. Additional site-specific reversions from arginine to lysine within the allR H2AX derivate are indicated. (<b>B</b>) H2AX-allR and acidic patch mutation E92A reduces H2AXub. WT or E92A H2AX/H2A constructs were transfected into HEK293T cells and analyzed by western blotting with the indicated antibodies. Arrows indicate ub forms. (SFB = S-tag, Flag epitope tag, and streptavidin-binding peptide tag; e = endogenous). Molecular weights (kDa) are indicated on the left of each panel. HEK293T cells were used for all cellular assays. (<b>C</b>) H2AX-K13/15 dependent ubiquitination requires the acidic patch. H2AX and derivatives were expressed in HEK293T cells (−) or (+) ionizing radiation (IR, 20 Gy). Samples were analyzed as in A 6 h post-IR treatment. (<b>D</b>) H2AX-K13/15 and K118/119-dependent ubiquitination requires the acidic patch. Cells were co-transfected with H2AX and derivatives along with Myc-RNF168 and analyzed as in C. (<b>E</b>) Phospho-competent H2AX S139 is not required <i>in cis</i> for H2AX K13/15ub. Cells were analyzed as in C. tub = tubulin loading control.</p

The nucleosome acidic patch is required <i>in vivo</i> for the DDR in human cells.

<p>(<b>A and B</b>) <i>In vivo</i> expression of the acidic patch interacting portion of LANA (1–32 amino acids) reduces 53BP1, but not MDC1, IRIF (ionizing radiation induced-foci). Human U2OS cells were transfected with GFP-LANA (1–32a.a.) followed by 2 Gy IR-treatment. Cells were analyzed by IF with the indicated antibodies 2 h post-IR. Representative IF images are shown. Nuclear DNA was visualized by Hoechst 33342 staining. Quantification of A is shown in B. 53BP1 and MDC1 IRIF were counted and graphed for cells (−) or (+) GFP-LANA (1–32a.a.). N = 3, >100 cells analyzed/experiment, error bars = SEM. Student's t-tests (paired) were performed and results indicated. *** = p-value<0.001, ns = not significant (i.e. p-value>0.05). (<b>C</b>) IF analysis of DDR factor foci formation after IR treatment in GFP-LANA and mutant GFP-LANA-8LRS10 expressing cells. Cells were treated with 2 Gy IR and processed for IF 2 h post-IR. IF analysis was performed as in A. (<b>D</b>) Quantification of 53BP1 IRIF from C. Graph represents values obtained from two independent experiments where foci from >100 cells were scored for GFP-LANA-8LRS10 expressing cells and non-GFP expressing cells. Error bars = SEM. Statistical analysis was performed as in B. (E) GFP-LANA (1–32a.a.) impairs recruitment of 53BP1 to laser damage. U2OS cells were transfected with GFP-LANA (1–32a.a.) followed by laser micro-irradiation. Cells were fixed and stained with antibodies as indicated 2 h post-laser damage. Quantification of 53BP1 and MDC1 laser lines were obtained from >50 damaged cells from two independent experiments. Error bars = SEM.</p

RING1B/BMI1- and RNF168-dependent ubiquitination of H2AX/H2A requires the nucleosome acidic patch <i>in vitro</i>.

<p>(<b>A</b>) Schematic for <i>in vitro</i> reconstitution of nucleosome core particles (NCPs). (<b>B</b>) Bacterially expressed and purified human histones. Histones were expressed, purified and reconstituted as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004178#s3" target="_blank">methods</a>. (<b>C</b>) Analysis of <i>in vitro</i> reconstituted NCPs. The 147 bp 601 DNA fragment was analyzed alone or after NCP reconstitution. DNA ladder indicates size (bp). (<b>D and E</b>) RING1B/BMI1 and RNF168 readily ubiquitinate H2AX within WT NCPs but not NCPs containing a mutation in the acidic patch (H2AX-E92A). In vitro Ub assays (4 h) were performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004178#s3" target="_blank">methods</a>. (<b>F</b>) RNF168 ubiquitinates WT H2AX and H2AX-E92A similarly when assayed in the context of free histones. Assays were performed as in E except with free histones and reactions were performed overnight. (<b>G and H</b>) RING1B/BMI1 and RNF168 readily ubiquitinate H2A within WT NCPs but not NCPs containing a mutation in the acidic patch (H2A-E92A). Experiments were performed as in D and E using H2A. (<b>I</b>) RNF168 ubiquitinates free WT H2A and H2A-E92A similarly. Experiments performed as in F.</p