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1.
Annu Rev Biochem ; 87: 263-294, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29709199

RESUMO

Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , Proteína Homóloga a MRE11/metabolismo , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunidade Inata , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/genética , Modelos Biológicos , Modelos Moleculares , Transdução de Sinais , Telômero/metabolismo
2.
Mol Cell ; 83(13): 2258-2275.e11, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37369199

RESUMO

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.


Assuntos
Síndromes de Tricotiodistrofia , Animais , Camundongos , Íntrons/genética , Síndromes de Tricotiodistrofia/genética , RNA Nucleotidiltransferases/genética , Splicing de RNA
3.
Cell ; 162(4): 701-3, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26276624

RESUMO

DNA transposition plays key roles in genome diversity, pathogenesis, and evolution. Yet, structural and mechanistic information on transposition targeting and regulation is limited. Arias-Palomo and Berger now define the decameric organization of the AAA+ ATPase IstB, unveiling key insights into its targeting and regulation of IstA transposase activity.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Elementos de DNA Transponíveis
4.
Mol Cell ; 81(14): 2989-3006.e9, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34197737

RESUMO

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/genética , DNA/genética , Exonucleases/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , DNA Helicases/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oncogenes/genética , Fosforilação/genética , Regulação para Cima/genética
5.
Mol Cell ; 81(20): 4228-4242.e8, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34686315

RESUMO

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Núcleo Celular/enzimologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Neoplásico/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Núcleo Celular/genética , DNA Helicases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Metilação , Neoplasias/genética , Proteínas Nucleares/genética , Estruturas R-Loop , RNA Neoplásico/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Transcrição Gênica , Ubiquitinação
6.
Cell ; 155(7): 1448-50, 2013 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-24360270

RESUMO

TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation.


Assuntos
5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Dioxigenases , Humanos
7.
Nat Chem Biol ; 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38671223

RESUMO

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states.

8.
Cell ; 145(2): 198-211, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21496641

RESUMO

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.


Assuntos
Endonucleases Flap/química , Endonucleases Flap/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , DNA/metabolismo , Análise Mutacional de DNA , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por Substrato
9.
J Biol Chem ; 300(6): 107368, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38750793

RESUMO

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.


Assuntos
Diester Fosfórico Hidrolases , Humanos , Biologia Computacional/métodos , Cristalografia por Raios X , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Motivos de Ligação ao RNA/genética
10.
Nucleic Acids Res ; 51(3): 1019-1033, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36477609

RESUMO

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.


Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.


Assuntos
Reparo do DNA , Fator de Transcrição TFIIH , DNA/metabolismo , Dano ao DNA , DNA de Cadeia Simples , Endonucleases/metabolismo , Nucleotídeos , Fator de Transcrição TFIIH/metabolismo
11.
Nucleic Acids Res ; 51(18): 9920-9937, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37665033

RESUMO

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.


Assuntos
Adenosina Trifosfatases , DNA Polimerase teta , Neoplasias , Novobiocina , Humanos , Adenosina Trifosfatases/metabolismo , Replicação do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Novobiocina/farmacologia
12.
Proc Natl Acad Sci U S A ; 119(34): e2207408119, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35969784

RESUMO

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.


Assuntos
Reparo do DNA , Proteína de Replicação A , Proteína de Xeroderma Pigmentoso Grupo A , DNA/metabolismo , Dano ao DNA , Ligação Proteica , Domínios Proteicos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
13.
Cell ; 139(1): 87-99, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19804755

RESUMO

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT(1)-BRCT(2) domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a bipartite MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Reparo do DNA , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11 , Modelos Moleculares , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
14.
Mol Cell ; 64(4): 688-703, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27871365

RESUMO

Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.


Assuntos
Proteínas de Caenorhabditis elegans/química , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Schizosaccharomyces pombe/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Cisplatino/química , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/efeitos da radiação , Formaldeído/química , Células HeLa , Humanos , Cinética , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
15.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34815340

RESUMO

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.


Assuntos
Sítios Frágeis do Cromossomo/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Linhagem Celular , Fragilidade Cromossômica/genética , Fragilidade Cromossômica/fisiologia , DNA/genética , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Variação Genética/genética , Instabilidade Genômica/genética , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo
16.
Dev Dyn ; 252(7): 1046-1060, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37002899

RESUMO

BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.


Assuntos
Colágeno Tipo IV , Peixe-Zebra , Animais , Humanos , Colágeno Tipo IV/genética , Células Endoteliais , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , Membrana Basal/metabolismo
17.
Hum Genet ; 142(2): 245-274, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36344696

RESUMO

Whilst DNA repeat expansions cause numerous heritable human disorders, their origins and underlying pathological mechanisms are often unclear. We collated a dataset comprising 224 human repeat expansions encompassing 203 different genes, and performed a systematic analysis with respect to key topological features at the DNA, RNA and protein levels. Comparison with controls without known pathogenicity and genomic regions lacking repeats, allowed the construction of the first tool to discriminate repeat regions harboring pathogenic repeat expansions (DPREx). At the DNA level, pathogenic repeat expansions exhibited stronger signals for DNA regulatory factors (e.g. H3K4me3, transcription factor-binding sites) in exons, promoters, 5'UTRs and 5'genes but were not significantly different from controls in introns, 3'UTRs and 3'genes. Additionally, pathogenic repeat expansions were also found to be enriched in non-B DNA structures. At the RNA level, pathogenic repeat expansions were characterized by lower free energy for forming RNA secondary structure and were closer to splice sites in introns, exons, promoters and 5'genes than controls. At the protein level, pathogenic repeat expansions exhibited a preference to form coil rather than other types of secondary structure, and tended to encode surface-located protein domains. Guided by these features, DPREx ( http://biomed.nscc-gz.cn/zhaolab/geneprediction/# ) achieved an Area Under the Curve (AUC) value of 0.88 in a test on an independent dataset. Pathogenic repeat expansions are thus located such that they exert a synergistic influence on the gene expression pathway involving inter-molecular connections at the DNA, RNA and protein levels.


Assuntos
Expansão das Repetições de DNA , DNA , Humanos , Íntrons/genética , RNA , Expansão das Repetições de Trinucleotídeos
18.
Cell ; 135(7): 1169-71, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19109888

RESUMO

Packaging of viral genomes into virus capsids requires powerful motors to overcome the repulsive force that builds as the nucleic acids are compressed. Through structural analyses of the T4 bacteriophage packaging motor gp17, Sun et al. (2008) now propose a packaging mechanism in which electrostatic forces cause the motor to alternate between tensed and relaxed conformational states.


Assuntos
Bacteriófago T4/metabolismo , Empacotamento do DNA , Proteínas Virais/química , Proteínas Virais/metabolismo , Montagem de Vírus
19.
Cell ; 133(5): 789-800, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18510924

RESUMO

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/genética , Sulfolobus acidocaldarius/enzimologia , Proteína Grupo D do Xeroderma Pigmentoso/química , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteínas Arqueais/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
20.
Cell ; 135(1): 97-109, 2008 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-18854158

RESUMO

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).


Assuntos
Proteínas Arqueais/metabolismo , Reparo do DNA , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Pyrococcus furiosus/química , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cristalografia por Raios X , DNA/química , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Dimerização , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Modelos Moleculares , Espalhamento a Baixo Ângulo , Schizosaccharomyces/genética , Técnicas do Sistema de Duplo-Híbrido , Difração de Raios X
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