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In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70–Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

Rev1–Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiaeRev1–Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1–Polζ cooperativity, interacting directly with the DNA template–primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.

The bacterial cyclic oligonucleotide-based antiphage signaling system (CBASS) is similar to the cGAS–STING system in humans, containing an enzyme that synthesizes a cyclic nucleotide on viral infection and an effector that senses the second messenger for the antiviral response. Cap5, containing a SAVED domain coupled to an HNH DNA endonuclease domain, is the most abundant CBASS effector, yet the mechanism by which it becomes activated for cell killing remains unknown. We present here high-resolution structures of full-length Cap5 from Pseudomonas syringae(Ps) with second messengers. The key to PsCap5 activation is a dimer-to-tetramer transition, whereby the binding of second messenger to dimer triggers an open-to-closed transformation of the SAVED domains, furnishing a surface for assembly of the tetramer. This movement propagates to the HNH domains, juxtaposing and converting two HNH domains into states for DNA destruction. These results show how Cap5 effects bacterial cell suicide and we provide proof-in-principle data that the CBASS can be extrinsically activated to limit bacterial infections.

As a core chromatin-regulatory scaffolding protein, WDR5 mediates numerous protein–protein interactions (PPIs) with other partner oncoproteins. However, small-molecule inhibitors that block these PPIs exert limited cell-killing effects. Here, we report structure–activity relationship studies in pancreatic ductal adenocarcinoma (PDAC) cells that led to the discovery of several WDR5 proteolysis-targeting chimer (PROTAC) degraders, including 11(MS132), a highly potent and selective von Hippel–Lindau (VHL)-recruiting WDR5 degrader, which displayed positive binding cooperativity between WDR5 and VHL, effectively inhibited proliferation in PDAC cells, and was bioavailable in mice and 25, a cereblon (CRBN)-recruiting WDR5 degrader, which selectively degraded WDR5 over the CRBN neo-substrate IKZF1. Furthermore, by conducting site-directed mutagenesis studies, we determined that WDR5 K296, but not K32, was involved in the PROTAC-induced WDR5 degradation. Collectively, these studies resulted in a highly effective WDR5 degrader, which could be a potential therapeutic for pancreatic cancer and several potentially useful tool compounds.

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I–DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17β-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I–DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)–mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells.

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40’s non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.

Emergence of SARS-CoV-2 coronavirus has led to millions of deaths globally. We present three high-resolution crystal structures of the SARS-CoV-2 nsp14 N7-methyltransferase core bound to S-adenosylmethionine (1.62 Å), S-adenosylhomocysteine (1.55 Å) and sinefungin (1.41 Å). We identify features of the methyltransferase core that are crucial for the development of antivirals and show SAH as the best scaffold for the design of antivirals against SARS-CoV-2 and other pathogenic coronaviruses.

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay—referred to as chromatin isolation by RNA–Cas13a complex—to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17β-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the SaccharomycescerevisiaePolζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31‚ Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein–protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiaePolδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe−4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA–dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation.

Focal facial dermal dysplasias (FFDDs) are rare genetic/developmental disorders characterised by bilateral ‘scar-like’ facial lesions. Four subtypes are classified by the bitemporal (FFDD1–3) or preauricular (FFDD4) lesion location. FFDD1–3 are differentiated by additional facial abnormalities and inheritance patterns. Although the genetic defects causing FFDD1 and FFDD2 remain unknown, recent studies identified defects causing FFDD3 and FFDD4. Here, the clinical phenotypes, genetic defects and inheritance of the four FFDD subtypes are described. In addition, the overlapping facial abnormalities in FFDD3 and two other genetic disorders, Ablepharon macrostomia syndrome and Barber-Say syndrome, are noted. Familiarity with the FFDDs by clinicians will further delineate the phenotypes and genetic/developmental defects of these dermal facial disorders.

The Zika virus (ZIKV) poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM) and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp). We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cβ atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.

DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.

DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ.

Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis–Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA–protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.

The development of inhibitors against Abl has changed the landscape for the treatment of chronic myelogenous leukemia (CML) and cancer in general. Beginning with the monumental discovery and approval of imatinib for CML, a second generation of inhibitors, nilotinib and dasatinib, has now gained approval for the treatment of CML. Notably, these second-generation inhibitors are active against many of the mutations in the Abl kinase that confer resistance to imatinib. However, resistance remains a major problem, and new inhibitors such as ponatinib and GNF2/GNF5 have been developed, with activity towards the common gatekeeper T315I mutation. We review here the mechanisms of Abl inhibition with an emphasis on structural elements that are important for the selectivity and design of new molecules. In particular, we focus on how changes in the conformation of the P-loop, the activation loop, the DFG motif, and other structural elements of Abl have been instrumental in developing an understanding of inhibitor binding.

Zika virus has emerged as a pathogen of major health concern. Here, we present a high-resolution (1.62-Å) crystal structure of the RNA helicase from the French Polynesia strain. The structure is similar to that of the RNA helicase from Dengue virus, with variability in the conformations of loops typically involved in binding ATP and RNA. We identify druggable 'hotspots' that are well suited for in silico and/or fragment-based high-throughput drug discovery.

Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3' untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8–10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3' Box B site than the 5' Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.

The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.

The amyloid hypothesis suggests that the process of amyloid-beta protein (Abeta) fibrillogenesis is responsible for triggering a cascade of physiological events that contribute directly to the initiation and progression of Alzheimer's disease. Consequently, preventing this process might provide a viable therapeutic strategy for slowing and/or preventing the progression of this devastating disease. A promising strategy to achieve prevention of this disease is to discover compounds that inhibit Abeta polymerization and deposition. Herein, we describe a new class of small molecules that inhibit Abeta aggregation, which is based on the chemical structure of apomorphine. These molecules were found to interfere with Abeta1-40 fibrillization as determined by transmission electron microscopy, Thioflavin T fluorescence and velocity sedimentation analytical ultracentrifugation studies. Using electron microscopy, time-dependent studies demonstrate that apomorphine and its derivatives promote the oligomerization of Abeta but inhibit its fibrillization. Preliminary structural activity studies demonstrate that the 10,11-dihydroxy substitutions of the D-ring of apomorphine are required for the inhibitory effectiveness of these aporphines, and methylation of these hydroxyl groups reduces their inhibitory potency. The ability of these small molecules to inhibit Abeta amyloid fibril formation appears to be linked to their tendency to undergo rapid autoxidation, suggesting that autoxidation product(s) acts directly or indirectly on Abeta and inhibits its fibrillization. The inhibitory properties of the compounds presented suggest a new class of small molecules that could serve as a scaffold for the design of more efficient inhibitors of Abeta amyloidogenesis in vivo.

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5′ overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.

ABSTRACTThe unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 Å resolution, as a complement to our earlier work on the BglII–DNA complex. Unexpectedly, the enzyme opens by a dramatic `scissor-like' motion, accompanied by a complete rearrangement of the α-helices at the dimer interface. Moreover, within each monomer, a set of residues — a ‘lever’ — lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA.

Axis determination in early Drosophilaembryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchbackmRNA in complex with Nanos to suppress hunchbacktranslation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the DrosophilaPumilio protein that binds RNA. The crystals belong to the space group P63with unit cell dimensions of a= b= 94.5 Å, c= 228.9 Å, α = β = 90°, γ = 120° and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of α-helical secondary structure.

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5′-ATGAATCCATA-3′, yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 Å with synchrotron radiation. The cocrystals belong to the space group P212121with unit cell dimensions of a= 114.8 Å, b= 91.1 Å, c= 66.4 Å, α = 90°, β = 90°, γ = 90°. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA–protein complexes.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1.5 Å resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar α/β core, but in BglII, the core is augmented by a β-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein–DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Description

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI–DNA complex, determined in the presence of Mn2+and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.

© 1997 Federation of European Biochemical Societies.

FokI is a type IIs restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence. The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains. We report here cocrystals of the complete FokI enzyme (579 amino acids) bound to a 20-bp DNA fragment containing its recognition sequence. The complex is amongst the largest protein-DNA complexes to be crystallized, and required macroseeding techniques for optimal crystal growth. The cocrystals diffract to at least 2.8 Å in resolution and belong to space group P2 1with unit cell dimensions of a=67.9 Å, b=119.8 Å, c=69.1 Å, β=96.6°. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment. This paper reports the first cocrystals of a type IIs restriction endonuclease.

No relevant conflicts of interest to declare.

Mantle Cell Lymphoma (MCL) is a fatal subtype of Non-Hodgkins Lymphoma. MCL is characterized by cell cycle dysregulation due to Cyclin D1 (CCND1) overexpression. Murine models overexpressing CCND1 do not develop B-cell proliferation. The SOX11 transcription factor is overexpressed in the majority of nodal human MCL and has been implicated in MCL pathogenesis. We have developed a transgenic Eµ-SOX11-EGFP mouse model that overexpresses SOX11 with aberrant B cell proliferation, lymphadenopathy, spenomegaly and hepatomegaly phenocopying the characteristics of patients with MCL.

Interferon regulatory factor 1 (IRF-1) is an essential factor involved in the regulation of type I interferon (IFN) and IFN-inducible genes. The protein consists of 329 amino acids that are highly conserved from mouse to human. Similar to other transcription factors, the protein is modular in nature with a basic N-terminal region involved in DNA binding and an acidic C-terminal region required for activation. We report here the expression, purification and co-crystallization of the minimal N-terminal region of IRF-1 involved in DNA binding (amino acids 1–113) with a 13 bp DNA fragment from the IFN-β promoter. The crystals diffract to at least 3.0Åin resolution and belong to space group R3 with unit cell parameters of a= b= 84.8Å, c= 203.7Å.

Reddy: Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.

Oncogenic activation of RASvia point mutations occurs in more than 30% of all human cancers, including hematopoietic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Investigations to understand the critical biochemical and biological mechanisms of RAS function are at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play an essential role in oncogenic RAS function, inhibiting them constitutes an attractive and important therapeutic approach for myeloid neoplasias and other cancers. Rigosertib is a novel styryl benzyl sulfone, which is in a Phase III clinical trial (ONTIME) for MDS. Here, we delineate the way rigosertib interacts with the RBDs of several RAS effector proteins: RAF, the PI3K family of proteins and RalGDS. To identify residues in the B-RAF RBD that interact with rigosertib, we recorded a series of 15N-1H HSQC spectra of 15N-labeled B-RAF RBD with increasing concentration of rigosertib. Strikingly, the chemical shift perturbations caused by addition of rigosertib are localized to the very region of the B-RAF-RBD implicated in RAS binding, namely the beta1 and beta2 strands and alpha3 helix (Fig 1). Additionally, this cluster of residues with largest chemical shift perturbation contains many of the same residues involved in RAS binding, namely Ile156, Lys164, Arg166, Thr167, Val168, Ala184 and Met187. These key residues are conserved within RAF RBDs, suggesting that rigosertib would bind to similar regions of the A- and c-RAF RBDs. Next, we examined the binding of rigosertib and GTP-RAS to wild type and mutant forms of c-RAF RBD that harbor mutations in residues that mediate binding to rigosertib. Our studies show that all mutations that cause dissociation of GTP-RAS binding also inhibit rigosertib binding to these mutant proteins. Taken together, the chemical shift data and mutagenesis data provide powerful evidence that rigosertib binds the B-RAF RBD at the same location as the RAS switch I region.

The type II restriction endonuclease BamHI has been expressed In E. coli, producing 100-fold more enzyme than the wild type Bacillus amyloliquefaciens H strain. This high yield has facilitated purification to homogeneity of large amounts of the enzyme, along with its crystallization in a form which diffracts to at least 1.9 A in X-ray analysis.