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1.
Biomol NMR Assign ; 18(1): 105-109, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38689205

RESUMO

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.


Assuntos
Mycobacterium tuberculosis , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/química , DNA Ligases/metabolismo
2.
J Biol Chem ; 300(5): 107216, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38522520

RESUMO

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.


Assuntos
DNA Ligase Dependente de ATP , DNA , Ribonucleotídeos , Humanos , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/química , DNA/metabolismo , DNA/química , Ribonucleotídeos/metabolismo , Ribonucleotídeos/química , Cristalografia por Raios X , Reparo do DNA
3.
J Mol Biol ; 436(4): 168410, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38135179

RESUMO

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) ß to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polß and LIGIIIα. Yet, the impact of the polß mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polß colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polß by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polß variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polß/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Ligase Dependente de ATP , DNA Polimerase beta , Reparo por Excisão , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Humanos , DNA Ligase Dependente de ATP/química , DNA Polimerase beta/química , Ligação Proteica , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
4.
Protein Sci ; 30(9): 1735-1756, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34056803

RESUMO

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIVBRCT -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.


Assuntos
DNA Ligase Dependente de ATP/química , Enzimas Reparadoras do DNA/química , DNA de Neoplasias/química , Proteína Quinase Ativada por DNA/química , Proteínas de Ligação a DNA/química , Autoantígeno Ku/química , Neoplasias/genética , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Cinética , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato
5.
Nucleic Acids Res ; 49(3): 1619-1630, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33444456

RESUMO

Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg2+ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg2+ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg2+ levels.


Assuntos
DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Mutação , Doenças da Imunodeficiência Primária/genética , Sítios de Ligação , DNA/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Humanos , Ligantes , Magnésio/química , Modelos Moleculares , Dobramento de Proteína , Síndrome
6.
Nucleic Acids Res ; 49(1): 306-321, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330937

RESUMO

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.


Assuntos
DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , DNA Ligase Dependente de ATP/química , Dimerização , Humanos , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos , Mutação , Mutação de Sentido Incorreto , Coloração Negativa , Mutação Puntual , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/química , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
7.
Nucleic Acids Res ; 47(14): 7147-7162, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31312841

RESUMO

DNA ligases join adjacent 5' phosphate (5'P) and 3' hydroxyl (3'OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase-DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3'OH terminus for nucleophilic attack on the 5'P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.


Assuntos
Proteínas de Bactérias/química , DNA Ligase Dependente de ATP/química , DNA/química , Metais/química , Prochlorococcus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Cinética , Manganês/química , Manganês/metabolismo , Metais/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Prochlorococcus/genética , Ligação Proteica , Domínios Proteicos
8.
Sci Adv ; 5(7): eaaw0590, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31334349

RESUMO

Inspired by the dynamics of the dissipative self-assembly of microtubules, chemically fueled synthetic systems with transient lifetimes are emerging for nonequilibrium materials design. However, realizing programmable or even adaptive structural dynamics has proven challenging because it requires synchronization of energy uptake and dissipation events within true steady states, which remains difficult to orthogonally control in supramolecular systems. Here, we demonstrate full synchronization of both events by ATP-fueled activation and dynamization of covalent DNA bonds via an enzymatic reaction network of concurrent ligation and cleavage. Critically, the average bond ratio and the frequency of bond exchange are imprinted into the energy dissipation kinetics of the network and tunable through its constituents. We introduce temporally and structurally programmable dynamics by polymerization of transient, dynamic covalent DNA polymers with adaptive steady-state properties in dependence of ATP fuel and enzyme concentrations. This approach enables generic access to nonequilibrium soft matter systems with adaptive and programmable dynamics.


Assuntos
Trifosfato de Adenosina/química , DNA/química , Microtúbulos/química , DNA Ligase Dependente de ATP/química , Endonucleases/química , Cinética , Simulação de Dinâmica Molecular , Fenômenos Físicos , Polímeros/química
9.
Biomol NMR Assign ; 13(2): 305-308, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31093909

RESUMO

In mammalian cells, the process of DNA ligation is necessary during DNA replication to create an intact "lagging" strand from a series of smaller Okazaki fragments and to repair DNA strand breaks that arise either due to the direct action of a DNA damaging agent or as a consequence of DNA damage excision during DNA repair. In humans, there are three genes that encode for members of the DNA ligase family (LIG1, LIG3 and LIG4) (Ellenberger and Tomkinson in Ann Rev Biochem 77:313-338. 2008). Although these genes code for polypeptides with overlapping functions in the nucleus, the only mitochondrial DNA ligase (DNA ligase IIIα), which is essential for mitochondrial genome maintenance, is encoded by the LIG3 gene (Lakshmipathy and Campbell in Mol Cell Biol 19:3869-3876, 1999; Zong et al. in Mol Cell 61:667-676, 2016) Because mitochondria play a central and multifunctional role in malignant tumor progression, there is emerging interest in targeting key mitochondrial proteins. Notably, there is evidence in pre-clinical models that inhibitors of DNA ligase IIIα, which is frequently up-regulated in cancer, preferentially target cancer cells via their effect on mitochondria (Zong et al. 2016). Since NMR spectroscopy provides unique capabilities for identifying small molecules that bind specifically to DNA ligase IIIα versus the other DNA ligases), the backbone 1HN, 13C, and 15N NMR resonance assignments were completed for a 222 amino acid DNA-binding domain of human DNA ligase III. These NMR assignments represent a vital first step towards developing DNA ligase III-selective inhibitors.


Assuntos
DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , DNA/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Humanos , Domínios Proteicos
10.
Structure ; 27(3): 485-496.e7, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30639225

RESUMO

The protein UHRF1 is crucial for DNA methylation maintenance. The tandem Tudor domain (TTD) of UHRF1 binds histone H3K9me2/3 with micromolar affinity, as well as unmethylated linker regions within UHRF1 itself, causing auto-inhibition. Recently, we showed that a methylated histone-like region of DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1 TTD with nanomolar affinity, permitting UHRF1 recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a LIG1K126me3 peptide. The data explain the basis for the high TTD-binding affinity of LIG1K126me3 and reveal that the interaction may be regulated by phosphorylation. Binding of LIG1K126me3 switches the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition is relieved. Our results provide structural insight into how UHRF1 performs its key function in epigenetic maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Arginina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Epigênese Genética , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos
11.
Nucleic Acids Res ; 47(3): 1428-1439, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30590734

RESUMO

Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central GTP-dependent polynucleotide kinase (KIN) domains that heal the broken ends to generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain (LIG). Here we report crystal structures of the Trl1-LIG domain from Chaetomium thermophilum at two discrete steps along the reaction pathway: the covalent LIG-(lysyl-Nζ)-AMP•Mn2+ intermediate and a LIG•ATP•(Mn2+)2 Michaelis complex. The structures highlight a two-metal mechanism whereby a penta-hydrated metal complex stabilizes the transition state of the ATP α phosphate and a second metal bridges the ß and γ phosphates to help orient the pyrophosphate leaving group. A LIG-bound sulfate anion is a plausible mimetic of the essential RNA terminal 2'-PO4. Trl1-LIG has a distinctive C-terminal domain that instates fungal Trl1 as the founder of an Rnl6 clade of ATP-dependent RNA ligase. We discuss how the Trl1-LIG structure rationalizes the large body of in vivo structure-function data for Saccharomyces cerevisiae Trl1.


Assuntos
Chaetomium/química , DNA Ligase Dependente de ATP/química , Diester Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo Ligases/química , Relação Estrutura-Atividade , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Domínio Catalítico , Chaetomium/enzimologia , Cristalografia por Raios X , DNA Ligase Dependente de ATP/genética , Metais/química , Diester Fosfórico Hidrolases/genética , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo Ligases/genética , Conformação Proteica , Domínios Proteicos , Splicing de RNA/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia
12.
FEBS J ; 285(21): 3959-3976, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30230716

RESUMO

Nonhomologous DNA end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV-dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C18 H14 N4 OS), whereas its oxidized form, SCR7-pyrazine, possesses a different molecular formula (C18 H12 N4 OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase-mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7-cyclized. Both forms blocked NHEJ in a Ligase IV-dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7-cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV-null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV-dependent manner, although SCR7-pyrazine is less specific to Ligase IV inside the cell.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , Neoplasias/patologia , Pirimidinas/farmacologia , Bases de Schiff/farmacologia , Morte Celular/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oxirredução , Recombinação V(D)J
13.
Nucleic Acids Res ; 46(19): 10474-10488, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30169742

RESUMO

DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.


Assuntos
DNA Ligase Dependente de ATP/química , DNA Ligases/química , DNA/química , Conformação de Ácido Nucleico , Domínios Proteicos , Archaea/enzimologia , Archaea/genética , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice
14.
Nat Commun ; 9(1): 2642, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29980672

RESUMO

DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states-an open lysyl-AMP intermediate, and a closed DNA-adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome.


Assuntos
Biocatálise , Domínio Catalítico , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , DNA/metabolismo , Adenina/metabolismo , Sequência de Bases , DNA Ligase Dependente de ATP/genética , Humanos , Lisina/metabolismo , Mutagênese/genética , Mutação/genética , Polimorfismo Genético , Ligação Proteica , Especificidade por Substrato
15.
Methods Mol Biol ; 1764: 449-473, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29605933

RESUMO

Small-angle X-ray scattering (SAXS) is an increasingly common and useful technique for structural characterization of molecules in solution. A SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. SAXS profiles can be utilized in a variety of molecular modeling applications, such as comparing solution and crystal structures, structural characterization of flexible proteins, assembly of multi-protein complexes, and modeling of missing regions in the high-resolution structure. Here, we describe protocols for modeling atomic structures based on SAXS profiles. The first protocol is for comparing solution and crystal structures including modeling of missing regions and determination of the oligomeric state. The second protocol performs multi-state modeling by finding a set of conformations and their weights that fit the SAXS profile starting from a single-input structure. The third protocol is for protein-protein docking based on the SAXS profile of the complex. We describe the underlying software, followed by demonstrating their application on interleukin 33 (IL33) with its primary receptor ST2 and DNA ligase IV-XRCC4 complex.


Assuntos
Modelos Moleculares , Simulação de Dinâmica Molecular , Espalhamento a Baixo Ângulo , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/química , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/química , Interleucina-33/metabolismo , Conformação Proteica , Software , Difração de Raios X
16.
Cell Rep ; 20(12): 2810-2819, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930678

RESUMO

Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Sequência de Aminoácidos , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Ligase Dependente de ATP/química , Humanos , Modelos Biológicos , Radiação Ionizante
17.
J Am Chem Soc ; 139(35): 12219-12227, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28780862

RESUMO

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Cristalografia por Raios X , DNA Ligase Dependente de ATP/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
18.
Mol Cell ; 67(4): 550-565.e5, 2017 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-28803780

RESUMO

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Metilação de DNA , Replicação do DNA , DNA/biossíntese , Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/genética , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Células-Tronco Embrionárias/enzimologia , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Lisina , Metilação , Camundongos , Modelos Moleculares , Mimetismo Molecular , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transfecção , Domínio Tudor , Ubiquitina-Proteína Ligases
19.
J Biol Chem ; 292(38): 15870-15879, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28751376

RESUMO

Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The LIG3 gene encodes a ubiquitous isoform in all tissues (LIG3α) and a germ line-specific splicing isoform (LIG3ß) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3α and LIG3ß and compared this framework to that of LIG1, the nuclear replicative ligase. The kinetic parameters of the LIG3 isoforms are nearly identical under all tested conditions, indicating that the BRCA1 C terminal (BRCT) domain specific to LIG3α does not alter ligation kinetics. Although LIG3 is only 22% identical to LIG1 across their conserved domains, the two enzymes had very similar maximal ligation rates. Comparison of the rate and equilibrium constants for LIG3 and LIG1 nevertheless revealed important differences. The LIG3 isoforms were seven times more efficient than LIG1 at ligating nicked DNA under optimal conditions, mainly because of their lower Km value for the DNA substrate. This could explain why LIG3 is less prone to abortive ligation than LIG1. Surprisingly, the affinity of LIG3 for Mg2+ was ten times weaker than that of LIG1, suggesting that Mg2+ availability regulates DNA ligation in vivo, because Mg2+ levels are higher in the mitochondria than in the nucleus. The biochemical differences between the LIG3 isoforms and LIG1 identified here will guide the understanding of both unique and overlapping biological roles of these critical enzymes.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Monofosfato de Adenosina/metabolismo , Sequência Conservada , DNA Ligase Dependente de ATP/química , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Magnésio/farmacologia , Modelos Moleculares , Conformação Proteica , Processamento de Proteína Pós-Traducional , Especificidade por Substrato
20.
J Biol Chem ; 292(34): 13914-13924, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28696258

RESUMO

In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases, and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5' and 3' overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3' overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3' overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5' overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3' overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells.


Assuntos
DNA Ligase Dependente de ATP/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Modelos Moleculares , Reparo de DNA por Recombinação , Recombinação V(D)J , Animais , Linhagem Celular , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Células HeLa , Humanos , Cinética , Mariposas , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , Especificidade por Substrato
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