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1.
Nucleic Acids Res ; 49(16): 9294-9309, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387694

RESUMO

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

2.
Nucleic Acids Res ; 49(16): 9310-9326, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387696

RESUMO

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

3.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
4.
Cell Death Dis ; 12(2): 165, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558481

RESUMO

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Tolerância a Radiação , Neoplasias da Bexiga Urinária/radioterapia , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Especificidade por Substrato , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
5.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199508

RESUMO

Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517-1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional ß-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.


Assuntos
Domínio Catalítico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Cristalização , DNA/metabolismo , Dano ao DNA/genética , Inativação Gênica , Células HCT116 , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transfecção , Zinco/metabolismo
6.
Essays Biochem ; 64(5): 819-830, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33095241

RESUMO

Helicases are enzymes that use the energy derived from ATP hydrolysis to catalyze the unwinding of DNA or RNA. The RecQ family of helicases is conserved through evolution from prokaryotes to higher eukaryotes and plays important roles in various DNA repair pathways, contributing to the maintenance of genome integrity. Despite their roles as general tumor suppressors, there is now considerable interest in exploiting RecQ helicases as synthetic lethal targets for the development of new cancer therapeutics. In this review, we summarize the latest developments in the structural and mechanistic study of RecQ helicases and discuss their roles in various DNA repair pathways. Finally, we consider the potential to exploit RecQ helicases as therapeutic targets and review the recent progress towards the development of small molecules targeting RecQ helicases as cancer therapeutics.


Assuntos
Reparo do DNA , Neoplasias/terapia , RecQ Helicases/metabolismo , Instabilidade Genômica , Humanos , Neoplasias/genética
7.
DNA Repair (Amst) ; 95: 102941, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866775

RESUMO

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5'-to-3' exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the 'unhooking' step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Animais , Proteínas de Ciclo Celular/química , DNA/efeitos dos fármacos , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/química , Humanos , Conformação Proteica
8.
Life Sci Alliance ; 3(7)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32467316

RESUMO

The cohesin subunit STAG2 has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between STAG2 and its paralog STAG1 To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing STAG2-wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of STAG2-deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in STAG2-mutated tumors.

9.
J Biol Chem ; 295(10): 2948-2958, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31914405

RESUMO

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.


Assuntos
DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/química , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência
10.
Structure ; 27(8): 1316-1325.e6, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31204252

RESUMO

Ubiquitylation, the posttranslational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. The SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. Here, we present a structural model of the KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography. Remarkably, CUE1, a canonical CUE domain, recognizes a cluster of exposed hydrophobic and surrounding charged/amphipathic residues on KAP1, which are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data suggest that CUE domains may have a wider function than simply recognizing ubiquitin and the ubiquitin-fold.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteína 28 com Motivo Tripartido/química , Proteína 28 com Motivo Tripartido/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Ubiquitina/metabolismo
11.
Hum Mutat ; 40(5): 566-577, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30817846

RESUMO

There is still around 50% of the familial breast cancer (BC) cases with an undefined genetic cause, here we have used next-generation sequencing (NGS) technology to identify new BC susceptibility genes. This approach has led to the identification of RECQL5, a member of RECQL-helicases family, as a new BC susceptibility candidate, which deserves further study. We have used a combination of whole exome sequencing in a family negative for mutations in BRCA1/2 throughout (BRCAX), in which we found a probably deleterious variant in RECQL5, and targeted NGS of the complete coding regions and exon-intron boundaries of the candidate gene in 699 BC Spanish BRCAX families and 665 controls. Functional characterization and in silico inference of pathogenicity were performed to evaluate the deleterious effect of detected variants. We found at least seven deleterious or likely deleterious variants among the cases and only one in controls. These results prompt us to propose RECQL5 as a gene that would be worth to analyze in larger studies to explore its possible implication in BC susceptibility.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Predisposição Genética para Doença , RecQ Helicases/genética , RecQ Helicases/metabolismo , Processamento Alternativo , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Variação Genética , Humanos , Perda de Heterozigosidade , Família Multigênica , Linhagem , Sequenciamento Completo do Exoma
12.
Mol Cell ; 73(3): 621-638.e17, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554943

RESUMO

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/química
13.
J Biol Chem ; 293(33): 12862-12876, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29880640

RESUMO

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Modelos Moleculares , Complexos Multienzimáticos/química , RNA Mitocondrial/química , RNA de Transferência/química , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Cristalografia por Raios X , Humanos , Metiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Espalhamento a Baixo Ângulo
14.
Nucleic Acids Res ; 45(7): 4231-4243, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28100692

RESUMO

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.


Assuntos
Modelos Moleculares , RecQ Helicases/química , Domínio Catalítico , Humanos , Mutação , Conformação Proteica em alfa-Hélice , RecQ Helicases/genética , RecQ Helicases/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
15.
Mol Cell ; 64(4): 704-719, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27871366

RESUMO

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.


Assuntos
Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/química , Instabilidade Genômica , Sequência de Aminoácidos , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Formaldeído/química , Expressão Gênica , Humanos , Cinética , Mutação , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Síndrome , Raios Ultravioleta
16.
PLoS One ; 11(2): e0148762, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26910052

RESUMO

The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders.


Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
17.
Structure ; 23(12): 2319-2330, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26636256

RESUMO

DNA polymerase theta (Polθ) has been identified as a crucial alternative non-homologous end-joining factor in mammalian cells. Polθ is upregulated in a range of cancer cell types defective in homologous recombination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attractive target for cancer treatment. We present crystal structures of the helicase domain of human Polθ in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA-stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polθ exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polθ helicase domain in the context of the Polθ tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/química , Sequência de Aminoácidos , Domínio Catalítico , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica
18.
Nucleic Acids Res ; 43(22): 11047-60, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26582912

RESUMO

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-ß-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-ß-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.


Assuntos
Enzimas Reparadoras do DNA/química , DNA/metabolismo , Exodesoxirribonucleases/química , Proteínas Nucleares/química , Domínio Catalítico , Proteínas de Ciclo Celular , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
19.
J Biol Chem ; 290(22): 13692-709, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25866208

RESUMO

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.


Assuntos
Proteínas E1A de Adenovirus/química , Proteínas de Ligação a DNA/química , Dissulfetos/química , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Proto-Oncogênicas/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Cromatografia Líquida , DNA/química , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neoplasias/metabolismo , Oxirredução , Oxigênio/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
20.
Nucleic Acids Res ; 43(10): 5221-35, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25901030

RESUMO

Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.


Assuntos
RecQ Helicases/química , Difosfato de Adenosina/química , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , RecQ Helicases/genética , RecQ Helicases/metabolismo , Anticorpos de Domínio Único/química , Zinco/química
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