Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.

NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.

LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex.

The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double-strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional 'partially open' conformation using luminescence resonance energy transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with small-angle X-ray scattering data, which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.

A Survey of Reported Disease-Related Mutations in the MRE11-RAD50-NBS1 Complex.

The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease. Here, we discuss, in a structural and biochemical context, mutations in each of the three MRN components that have been associated with diseases such as ataxia telangiectasia-like disorder (ATLD), Nijmegen breakage syndrome (NBS), NBS-like disorder (NBSLD) and certain types of cancers. Overall, deepening our understanding of disease-causing mutations of the MRN complex at the structural and biochemical level is foundational to the future aim of treating diseases associated with these aberrations.

Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions.

Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11-Rad50-Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.

Adjacent mutations in the archaeal Rad50 ABC ATPase D-loop disrupt allosteric regulation of ATP hydrolysis through different mechanisms.

DNA damage is the driving force for mutation and genomic instability, which can both lead to cell death or carcinogenesis. DNA double strand breaks are detected and processed in part by the Mre11-Rad50-Nbs1 protein complex. Although the Mre11-Rad50-Nbs1 complex is essential, several spontaneous mutations have been noted in various cancers. One of these mutations, within a conserved motif of Rad50, resulted in an outlier curative response in a clinical trial. We show through biochemical and biophysical characterization that this cancer-associated mutation and a second mutation to the adjacent residue, previously described in a breast cancer patient, both have gain-of-function Rad50 ATP hydrolysis activity that results not from faster association of the ATP-bound form but faster dissociation leading to less stable Rad50 dimer. This disruption impairs the regulatory functions of the protein complex leading to a loss of exonuclease activity from Mre11. Interestingly, these two mutations affect Rad50 structure and dynamics quite differently. These studies describe the relationship between function, structure, and molecular motions in improperly regulated Rad50, which reveal the underlying biophysical mechanism for how these two cancer-associated mutations affect the cell.

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

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.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

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.

Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Mitosis inhibits DNA double-strand break repair to guard against telomere fusions.

Mitotic cells inactivate DNA double-strand break (DSB) repair, but the rationale behind this suppression remains unknown. Here, we unravel how mitosis blocks DSB repair and determine the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora B kinase-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. We conclude that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.

Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

Histone 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.

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.

A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice.

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

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.

Solution structure of tRNAVal from refinement of homology model against residual dipolar coupling and SAXS data.

A procedure is presented for refinement of a homology model of E. coli tRNA(Val), originally based on the X-ray structure of yeast tRNA(Phe), using experimental residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. A spherical sampling algorithm is described for refinement against SAXS data that does not require a globbic approximation, which is particularly important for nucleic acids where such approximations are less appropriate. Substantially higher speed of the algorithm also makes its application favorable for proteins. In addition to the SAXS data, the structure refinement employed a sparse set of NMR data consisting of 24 imino N-H(N) RDCs measured with Pf1 phage alignment, and 20 imino N-H(N) RDCs obtained from magnetic field dependent alignment of tRNA(Val). The refinement strategy aims to largely retain the local geometry of the 58% identical tRNA(Phe) by ensuring that the atomic coordinates for short, overlapping segments of the ribose-phosphate backbone and the conserved base pairs remain close to those of the starting model. Local coordinate restraints are enforced using the non-crystallographic symmetry (NCS) term in the XPLOR-NIH or CNS software package, while still permitting modest movements of adjacent segments. The RDCs mainly drive the relative orientation of the helical arms, whereas the SAXS restraints ensure an overall molecular shape compatible with experimental scattering data. The resulting structure exhibits good cross-validation statistics (jack-knifed Q (free) = 14% for the Pf1 RDCs, compared to 25% for the starting model) and exhibits a larger angle between the two helical arms than observed in the X-ray structure of tRNA(Phe), in agreement with previous NMR-based tRNA(Val) models.

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 Ca(2+), Mg(2+), Mn(2+), and Sr(2+) 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.

Efficient ligation of the Schistosoma hammerhead ribozyme.

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165.

Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2'fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF(165) aptamer binds specifically to the VEGF(165) isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF(165)-aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF(165) for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD-aptamer complex are present in the full-length VEGF(164)-aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF(165)-aptamer complex.

Fast cleavage kinetics of a natural hammerhead ribozyme.

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.