Publications by authors named "Dinshaw J Patel"

255 Publications

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

Nat Commun 2021 05 3;12(1):2490. Epub 2021 May 3.

Department of Biochemistry, University of California, Riverside, CA, USA.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1). Engagement of DNMT1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.
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http://dx.doi.org/10.1038/s41467-021-22665-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093215PMC
May 2021

Integrative analysis reveals unique structural and functional features of the Smc5/6 complex.

Proc Natl Acad Sci U S A 2021 May;118(19)

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.
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http://dx.doi.org/10.1073/pnas.2026844118DOI Listing
May 2021

DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network.

Mol Cell 2021 Apr 8. Epub 2021 Apr 8.

Novo Nordisk Foundation Center for Protein Research (CPR), University of Copenhagen, Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark. Electronic address:

From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.
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http://dx.doi.org/10.1016/j.molcel.2021.03.041DOI Listing
April 2021

Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides.

J Biol Chem 2021 Mar 26:100600. Epub 2021 Mar 26.

Hormel Institute, University of Minnesota, 801 16(th) Ave NE, Austin, MN, 55912. Electronic address:

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanism(s) used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET), and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain, PI-(4,5)P which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically-relevant PI-(4,5)P levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.
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http://dx.doi.org/10.1016/j.jbc.2021.100600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091061PMC
March 2021

Oligomeric quaternary structure of Escherichia coli and Mycobacterium smegmatis Lhr helicases is nucleated by a novel C-terminal domain composed of five winged-helix modules.

Nucleic Acids Res 2021 04;49(7):3876-3887

Molecular Biology and Structural Biology Programs, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Mycobacterium smegmatis Lhr (MsmLhr; 1507-aa) is the founder of a novel clade of bacterial helicases. MsmLhr consists of an N-terminal helicase domain (aa 1-856) with a distinctive tertiary structure (Lhr-Core) and a C-terminal domain (Lhr-CTD) of unknown structure. Here, we report that Escherichia coli Lhr (EcoLhr; 1538-aa) is an ATPase, translocase and ATP-dependent helicase. Like MsmLhr, EcoLhr translocates 3' to 5' on ssDNA and unwinds secondary structures en route, with RNA:DNA hybrid being preferred versus DNA:DNA duplex. The ATPase and translocase activities of EcoLhr inhere to its 877-aa Core domain. Full-length EcoLhr and MsmLhr have homo-oligomeric quaternary structures in solution, whereas their respective Core domains are monomers. The MsmLhr CTD per se is a homo-oligomer in solution. We employed cryo-EM to solve the structure of the CTD of full-length MsmLhr. The CTD protomer is composed of a series of five winged-helix (WH) modules and a β-barrel module. The CTD adopts a unique homo-tetrameric quaternary structure. A Lhr-CTD subdomain, comprising three tandem WH modules and the β-barrel, is structurally homologous to AlkZ, a bacterial DNA glycosylase that recognizes and excises inter-strand DNA crosslinks. This homology is noteworthy given that Lhr is induced in mycobacteria exposed to the inter-strand crosslinker mitomycin C.
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http://dx.doi.org/10.1093/nar/gkab145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053096PMC
April 2021

Purification of Cytosolic Phospholipase Aα C2-domain after Expression in Soluble Form in .

Bio Protoc 2021 Feb 5;11(3):e3906. Epub 2021 Feb 5.

Hormel Institute, University of Minnesota, Austin, MN, U.S.A.

Previous expression/purification strategies for cytosolic phospholipase Aα C2-domain in have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase Aα C2-domain in soluble form in and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase Aα ( Hirano , 2019 ).
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http://dx.doi.org/10.21769/BioProtoc.3906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952920PMC
February 2021

DNA-driven condensation assembles the meiotic DNA break machinery.

Nature 2021 04 17;592(7852):144-149. Epub 2021 Mar 17.

Molecular Biology Program, Memorial Sloan Kettering Cancer Center and Howard Hughes Medical Institute, New York, New York, USA.

The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein. The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures, but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery. Each subcomplex of Rec114-Mei4 (a 2:1 heterotrimer) or Mer2 (a coiled-coil-containing homotetramer) is monodispersed in solution, but they independently condense with DNA into reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive this condensation. Mutations that weaken protein-DNA interactions strongly disrupt both condensate formation and DSBs in vivo, and thus these processes are highly correlated. In vitro, condensates fuse into mixed RMM clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centres of DSB activity. We propose that multilayered control of Spo11 arises from the recruitment of regulatory components and modulation of the biophysical properties of the condensates.
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http://dx.doi.org/10.1038/s41586-021-03374-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016751PMC
April 2021

Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex.

Proc Natl Acad Sci U S A 2021 Feb;118(8)

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.
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http://dx.doi.org/10.1073/pnas.2024512118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923543PMC
February 2021

Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex.

Genes Dev 2021 Mar 11;35(5-6):392-409. Epub 2021 Feb 11.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), 1030 Vienna, Austria.

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in , facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
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http://dx.doi.org/10.1101/gad.347989.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919418PMC
March 2021

Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.

Mol Cell 2021 03 19;81(5):1100-1115.e5. Epub 2021 Jan 19.

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China. Electronic address:

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13a in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
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http://dx.doi.org/10.1016/j.molcel.2020.12.033DOI Listing
March 2021

The Card1 nuclease provides defence during type III CRISPR immunity.

Nature 2021 02 18;590(7847):624-629. Epub 2021 Jan 18.

Laboratory of Bacteriology, The Rockefeller University, New York, NY, USA.

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.
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http://dx.doi.org/10.1038/s41586-021-03206-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906951PMC
February 2021

Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases.

Nature 2021 02 23;590(7846):498-503. Epub 2020 Dec 23.

Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, China.

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36). However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.
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http://dx.doi.org/10.1038/s41586-020-03069-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889650PMC
February 2021

Structure-function analysis of microRNA 3'-end trimming by Nibbler.

Proc Natl Acad Sci U S A 2020 12 16;117(48):30370-30379. Epub 2020 Nov 16.

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065;

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of and Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'--methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'--methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
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http://dx.doi.org/10.1073/pnas.2018156117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720153PMC
December 2020

Keeping innate immune response in check: when cGAS meets the nucleosome.

Cell Res 2020 12;30(12):1055-1056

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA.

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http://dx.doi.org/10.1038/s41422-020-00423-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784697PMC
December 2020

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation.

Proc Natl Acad Sci U S A 2020 08 16;117(31):18439-18447. Epub 2020 Jul 16.

Department of Biochemistry, University of California, Riverside, CA 92521;

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.
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http://dx.doi.org/10.1073/pnas.2009316117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414182PMC
August 2020

A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.

Science 2020 07 28;369(6499):54-59. Epub 2020 May 28.

Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.

The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.
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http://dx.doi.org/10.1126/science.abb6151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7975689PMC
July 2020

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers.

RNA 2020 08 5;26(8):982-995. Epub 2020 May 5.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.
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http://dx.doi.org/10.1261/rna.075341.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373991PMC
August 2020

Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis.

PLoS Genet 2020 03 18;16(3):e1008422. Epub 2020 Mar 18.

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.
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http://dx.doi.org/10.1371/journal.pgen.1008422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105138PMC
March 2020

Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.

Cell Res 2020 02 10;30(2):182-184. Epub 2020 Jan 10.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

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http://dx.doi.org/10.1038/s41422-019-0272-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015049PMC
February 2020

Crucial Roles of Two Hydrated Mg Ions in Reaction Catalysis of the Pistol Ribozyme.

Angew Chem Int Ed Engl 2020 02 9;59(7):2837-2843. Epub 2020 Jan 9.

Institute of Organic Chemistry and Center for Molecular Biosciences, Leopold-Franzens University, Innrain 80-82, 6020, Innsbruck, Austria.

Pistol ribozymes constitute a new class of small self-cleaving RNAs. Crystal structures have been solved, providing three-dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage, corresponding to the pre-catalytic state, a vanadate mimic of the transition state, and the product. The results led to the proposed underlying chemical mechanism. Importantly, a hydrated Mg ion remains innersphere-coordinated to N7 of G33 in all three states, and is consistent with its likely role as acid in general acid base catalysis (δ and β catalysis). Strikingly, the new structures shed light on a second hydrated Mg ion that approaches the scissile phosphate from its binding site in the pre-cleavage state to reach out for water-mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg ion appears to be the stabilization of product conformation. This study delivers a mechanistic understanding of ribozyme-catalyzed backbone cleavage.
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http://dx.doi.org/10.1002/anie.201912522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027511PMC
February 2020

Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.

Proc Natl Acad Sci U S A 2019 12 18;116(49):24507-24516. Epub 2019 Nov 18.

Molecular Biology Program, Sloan Kettering Institute, New York, NY 10065;

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.
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http://dx.doi.org/10.1073/pnas.1913546116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900545PMC
December 2019

The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation.

Nat Struct Mol Biol 2019 08 5;26(8):720-731. Epub 2019 Aug 5.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria.

The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1-Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. Our data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly.
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http://dx.doi.org/10.1038/s41594-019-0270-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828549PMC
August 2019

CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA Cleavage with ApA>p Formation Terminating RNase Activity.

Mol Cell 2019 09 17;75(5):944-956.e6. Epub 2019 Jul 17.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cA) from ATP and subsequent cA-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA CARF-binding pocket involved in autoinhibitory regulation of RNase activity.
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http://dx.doi.org/10.1016/j.molcel.2019.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731128PMC
September 2019

Second Messenger cA Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its Release Path.

Mol Cell 2019 09 17;75(5):933-943.e6. Epub 2019 Jul 17.

Structural Biology Program Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA), and products (cA), to decipher mechanistic aspects of cA formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA intermediates and subsequent cA formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA signaling pathway.
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http://dx.doi.org/10.1016/j.molcel.2019.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731140PMC
September 2019

Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia.

Nat Commun 2019 06 19;10(1):2691. Epub 2019 Jun 19.

Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.
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http://dx.doi.org/10.1038/s41467-019-10523-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584500PMC
June 2019

Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation.

Proc Natl Acad Sci U S A 2019 06 29;116(24):11946-11955. Epub 2019 May 29.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

The cyclic GMP-AMP synthase (cGAS)-cGAMP-STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGAS-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGAS-DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS-DNA interface. We also report the crystal structure of the h-cGAS-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGAS, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.
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http://dx.doi.org/10.1073/pnas.1905013116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575157PMC
June 2019

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression.

Nat Commun 2019 05 21;10(1):2261. Epub 2019 May 21.

Laboratory for RNA Molecular Biology, The Rockefeller University, 1230 York Ave, Box 186, New York, NY, 10065, USA.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.
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http://dx.doi.org/10.1038/s41467-019-08620-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529454PMC
May 2019

Hatchet ribozyme structure and implications for cleavage mechanism.

Proc Natl Acad Sci U S A 2019 05 14;116(22):10783-10791. Epub 2019 May 14.

Life Science Institute, Zhejiang University, 310058 Hangzhou, China;

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.
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http://dx.doi.org/10.1073/pnas.1902413116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561176PMC
May 2019

Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase Aα.

Elife 2019 05 3;8. Epub 2019 May 3.

Hormel Institute, University of Minnesota, Austin, United States.

Ca-stimulated translocation of cytosolic phospholipase Aα (cPLAα) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLAα preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLAα C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl--glycero-3-phosphocholine (DHPC) and Ca ions. Two Ca are complexed at previously reported locations in the lipid-free C2-domain. One of these Caions, along with a third Ca, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLAα activity. The DHPC-binding mode of the cPLAα C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.
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http://dx.doi.org/10.7554/eLife.44760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550875PMC
May 2019

REC114 Partner ANKRD31 Controls Number, Timing, and Location of Meiotic DNA Breaks.

Mol Cell 2019 06 16;74(5):1053-1068.e8. Epub 2019 Apr 16.

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA. Electronic address:

Double-strand breaks (DSBs) initiate the homologous recombination that is crucial for meiotic chromosome pairing and segregation. Here, we unveil mouse ANKRD31 as a lynchpin governing multiple aspects of DSB formation. Spermatocytes lacking ANKRD31 have altered DSB locations and fail to target DSBs to the pseudoautosomal regions (PARs) of sex chromosomes. They also have delayed and/or fewer recombination sites but, paradoxically, more DSBs, suggesting DSB dysregulation. Unrepaired DSBs and pairing failures-stochastic on autosomes, nearly absolute on X and Y-cause meiotic arrest and sterility in males. Ankrd31-deficient females have reduced oocyte reserves. A crystal structure defines a pleckstrin homology (PH) domain in REC114 and its direct intermolecular contacts with ANKRD31. In vivo, ANKRD31 stabilizes REC114 association with the PAR and elsewhere. Our findings inform a model in which ANKRD31 is a scaffold anchoring REC114 and other factors to specific genomic locations, thereby regulating DSB formation.
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http://dx.doi.org/10.1016/j.molcel.2019.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555648PMC
June 2019