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1.
J Mol Biol ; 436(11): 168589, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38677494

RESUMO

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.


Assuntos
DNA Helicases , Proteínas MutL , DNA/química , DNA Helicases/química , DNA Helicases/genética , Proteínas MutL/química , Proteínas MutL/genética , Ligação Proteica , Domínios Proteicos , Mesilatos/química , Reagentes de Ligações Cruzadas/química
2.
Cancer Res ; 84(9): 1443-1459, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38359148

RESUMO

AXIN1 is a major component of the ß-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to ß-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3ß, ß-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper ß-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected ß-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on ß-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the ß-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect ß-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other ß-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its ß-catenin regulatory function, whereas the majority are passenger mutations.


Assuntos
Proteína Axina , Mutação de Sentido Incorreto , beta Catenina , Proteína Axina/genética , Proteína Axina/metabolismo , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Transdução de Sinais/genética , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Células HEK293 , Linhagem Celular Tumoral , Ligação Proteica
3.
Nat Struct Mol Biol ; 30(8): 1172-1182, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37460897

RESUMO

RNA-guided type V CRISPR-Cas12 effectors provide adaptive immunity against mobile genetic elements (MGEs) in bacteria and archaea. Among diverse Cas12 enzymes, the recently identified Cas12m2 (CRISPR-Cas type V-M) is highly compact and has a unique RuvC active site. Although the non-canonical RuvC triad does not permit dsDNA cleavage, Cas12m2 still protects against invading MGEs through transcriptional silencing by strong DNA binding. However, the molecular mechanism of RNA-guided genome inactivation by Cas12m2 remains unknown. Here we report cryo-electron microscopy structures of two states of Cas12m2-CRISPR RNA (crRNA)-target DNA ternary complexes and the Cas12m2-crRNA binary complex, revealing structural dynamics during crRNA-target DNA heteroduplex formation. The structures indicate that the non-target DNA strand is tightly bound to a unique arginine-rich cluster in the recognition (REC) domains and the non-canonical active site in the RuvC domain, ensuring strong DNA-binding affinity of Cas12m2. Furthermore, a structural comparison of Cas12m2 with TnpB, a putative ancestor of Cas12 enzymes, suggests that the interaction of the characteristic coiled-coil REC2 insertion with the protospacer-adjacent motif-distal region of the heteroduplex is crucial for Cas12m2 to engage in adaptive immunity. Collectively, our findings improve mechanistic understanding of diverse type V CRISPR-Cas effectors and provide insights into the evolution of TnpB to Cas12 enzymes.


Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Microscopia Crioeletrônica , Bactérias/metabolismo , RNA/metabolismo , DNA/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo
4.
Stem Cell Rev Rep ; 19(6): 2052-2072, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37266894

RESUMO

Self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) are carefully controlled by extrinsic and intrinsic factors, to ensure the lifelong process of hematopoiesis. Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein implicated in DNA repair and transcriptional regulation. Although previous studies have emphasized the necessity of studying APEX1 in a lineage-specific context and its role in progenitor differentiation, no studies have assessed the role of APEX1, nor its two enzymatic domains, in supporting adult HSPC function. In this study, we demonstrated that complete loss of APEX1 from murine bone marrow HSPCs (induced by CRISPR/Cas9) caused severe hematopoietic failure following transplantation, as well as a HSPC expansion defect in culture conditions maintaining in vivo HSC functionality. Using specific inhibitors against either the nuclease or redox domains of APEX1 in combination with single cell transcriptomics (CITE-seq), we found that both APEX1 nuclease and redox domains are regulating mouse HSPCs, but through distinct underlying transcriptional changes. Inhibition of the APEX1 nuclease function resulted in loss of HSPCs accompanied by early activation of differentiation programs and enhanced lineage commitment. By contrast, inhibition of the APEX1 redox function significantly downregulated interferon-stimulated genes and regulons in expanding HSPCs and their progeny, resulting in dysfunctional megakaryocyte-biased HSPCs, as well as loss of monocytes and lymphoid progenitor cells. In conclusion, we demonstrate that APEX1 is a key regulator for adult regenerative hematopoiesis, and that the APEX1 nuclease and redox domains differently impact proliferating HSPCs.


Assuntos
Endonucleases , Transplante de Células-Tronco Hematopoéticas , Animais , Camundongos , Endonucleases/metabolismo , Células-Tronco Hematopoéticas , Diferenciação Celular/genética , Oxirredução
6.
Mol Cell ; 82(23): 4487-4502.e7, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36427491

RESUMO

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.


Assuntos
Proteínas Associadas a CRISPR , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , Plasmídeos , RNA , RNA Guia de Cinetoplastídeos/metabolismo
7.
J Exp Med ; 219(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35947077

RESUMO

The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome.


Assuntos
Agamaglobulinemia , Transtorno do Espectro Autista , Animais , Transtorno do Espectro Autista/genética , Heterozigoto , Camundongos , Mutação/genética , Fator de Transcrição PAX5/genética
8.
Nucleic Acids Res ; 50(11): 6224-6234, 2022 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-35670670

RESUMO

DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100-1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides is removed from the daughter strand followed by re-synthesis. How these opposite activities are coordinated is poorly understood. Here we show that the Escherichia coli MutL protein binds to the 3' end of the resected strand and blocks access of Pol I and Pol III. The cryo-EM structure of an 85-kDa MutL-DNA complex, determined to 3.7 Å resolution, reveals a unique DNA binding mode that positions MutL at the 3' end of a primer-template, but not at a 5' resected DNA end or a blunt DNA end. Hence, our work reveals a novel role for MutL in the final stages of mismatch repair by preventing premature DNA synthesis during removal of the mismatched strand.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli , Proteínas MutL , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas MutL/genética
10.
Nat Cell Biol ; 23(6): 608-619, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34108662

RESUMO

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR-Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.


Assuntos
Dano ao DNA , Reparo do DNA , Instabilidade Genômica , Fator 1 de Elongação de Peptídeos/metabolismo , Elongação da Transcrição Genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Fator 1 de Elongação de Peptídeos/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Ubiquitinação
11.
J Mol Biol ; 433(15): 167058, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34023401

RESUMO

Rapidly spreading new variants of SARS-CoV-2 carry multiple mutations in the viral spike protein which attaches to the angiotensin converting enzyme 2 (ACE2) receptor on host cells. Among these mutations are amino acid changes N501Y (lineage B.1.1.7, first identified in the UK), and the combination N501Y, E484K, K417N (B.1.351, first identified in South Africa), all located at the interface on the receptor binding domain (RBD). We experimentally establish that RBD containing the N501Y mutation results in 7-fold stronger binding to the hACE2 receptor than wild type RBD. The E484K mutation only slightly enhances the affinity for the receptor, while K417N attenuates affinity. As a result, RBD from B.1.351 containing all three mutations binds 3-fold stronger to hACE2 than wild type RBD but 2-fold weaker than N501Y. However, the recently emerging double mutant E484K/N501Y binds even stronger than N501Y. The independent evolution of lineages containing mutations with different effects on receptor binding affinity, viral transmission and immune evasion underscores the importance of global viral genome surveillance and functional characterization.


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Sítios de Ligação , Células HEK293 , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , SARS-CoV-2/classificação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
12.
Nat Struct Mol Biol ; 28(4): 373-381, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33820992

RESUMO

DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.


Assuntos
DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Proteínas MutL/ultraestrutura , Proteína MutS de Ligação de DNA com Erro de Pareamento/ultraestrutura , Conformação Proteica , Microscopia Crioeletrônica , DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética
13.
Int J Mol Sci ; 21(18)2020 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-32937838

RESUMO

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Autoantígeno Ku/genética , Proteínas Nucleares/genética , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Proteína Quinase Ativada por DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Camundongos , Fosforilação/genética
14.
Sci Adv ; 6(33): eaba6617, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32851167

RESUMO

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.


Assuntos
Gliotoxina , Infecções por HIV , HIV-1 , Gliotoxina/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Células HeLa , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição/metabolismo
15.
Sci Adv ; 6(25): eaaz4849, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32596446

RESUMO

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.


Assuntos
Proteína 9 Associada à CRISPR , Campylobacter jejuni , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , DNA/genética , Edição de Genes , Humanos , RNA Guia de Cinetoplastídeos/genética
16.
Gastroenterology ; 158(4): 1029-1043.e10, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31857074

RESUMO

BACKGROUND & AIMS: The ß-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of ß-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. METHODS: Plasmids encoding tagged full-length ß-catenin (CTNNB1) or ß-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of ß-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of ß-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. RESULTS: Mice injected with plasmids encoding K335I or N387K ß-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT ß-catenin and MET. K335I and N387K ß-catenin bound APC with lower affinity than WT ß-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by ß-catenin. Studies of protein structures supported the observed changes in relative binding affinities. CONCLUSION: Expression of ß-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in ß-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of ß-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase ß-catenin signaling in cancer cells.


Assuntos
Carcinogênese/genética , Genes APC/fisiologia , Neoplasias Hepáticas/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Células HCT116 , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos , Mutação , Plasmídeos/farmacologia , Proteínas Proto-Oncogênicas c-met , Transcrição Gênica
17.
Nucleic Acids Res ; 47(22): 11667-11680, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31598722

RESUMO

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Pareamento Incorreto de Bases/genética , Proteína 9 Associada à CRISPR/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Endodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo
18.
Nat Chem Biol ; 14(7): 642-651, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29915237

RESUMO

Over the last decade, research on distinct types of CRISPR systems has revealed many structural and functional variations. Recently, several novel types of single-polypeptide CRISPR-associated systems have been discovered including Cas12a/Cpf1 and Cas13a/C2c2. Despite distant similarities to Cas9, these additional systems have unique structural and functional features, providing new opportunities for genome editing applications. Here, relevant fundamental features of natural and engineered CRISPR-Cas variants are compared. Moreover, practical matters are discussed that are essential for dedicated genome editing applications, including nuclease regulation and delivery, target specificity, as well as host repair diversity.


Assuntos
Sistemas CRISPR-Cas/genética , Exorribonucleases/genética , Edição de Genes , Exorribonucleases/metabolismo
19.
PLoS One ; 12(2): e0170762, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28234898

RESUMO

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Biossíntese de Proteínas/efeitos da radiação , Proteoma/efeitos da radiação , Proteômica , Animais , Linhagem Celular , Telefone Celular , Humanos , Camundongos , Transcriptoma/efeitos da radiação , Tecnologia sem Fio
20.
Nucleic Acids Res ; 45(8): 4507-4518, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28168276

RESUMO

The tumor suppressor BRCA2 is a large multifunctional protein mutated in 50-60% of familial breast cancers. BRCA2 interacts with many partners and includes multiple regions with potentially disordered structure. In homology directed DNA repair BRCA2 delivers RAD51 to DNA resulting in removal of RPA and assembly of a RAD51 nucleoprotein filament. Dynamic rearrangements of BRCA2 likely drive this molecular hand-off initiating DNA strand exchange. We show human BRCA2 forms oligomers which can have an extended shape. Scanning force microscopy and quantitative single molecule fluorescence define the variety of BRCA2 complexes, reveal dramatic rearrangements upon RAD51 binding and the loading of RAD51 patches on single strand DNA. At sites of repair in cell nuclei, super-resolution microscopy shows BRCA2 and RAD51 arranged in largely separate locations. We identified dynamic structural transitions in BRCA2 complexes suggested to facilitate loading of RAD51 onto RPA coated single strand DNA and subsequent release of BRCA2.


Assuntos
Proteína BRCA2/genética , Núcleo Celular/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sítios de Ligação , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Quebras de DNA de Cadeia Simples , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Microscopia de Força Atômica , Ligação Proteica , Multimerização Proteica , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Imagem Individual de Molécula
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