RESUMEN
In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.
Asunto(s)
Proteínas Portadoras/genética , Inestabilidad Cromosómica/genética , Proteínas de Unión al ADN/genética , Mitosis/genética , Proteínas Nucleares/genética , Transactivadores/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN/genética , Fase G1/genética , Genoma Humano/genética , Inestabilidad Genómica/genética , Histonas , Humanos , Fosforilación , Transducción de Señal/genéticaRESUMEN
Telomeres protect chromosome ends from unscheduled DNA repair, including from the MRN (MRE11, RAD50, NBS1) complex, which processes double-stranded DNA breaks (DSBs) via activation of the ATM kinase, promotes DNA end-tethering aiding the non-homologous end-joining (NHEJ) pathway, and initiates DSB resection through the MRE11 nuclease. A protein motif (MIN, for MRN inhibitor) inhibits MRN at budding yeast telomeres by binding to RAD50 and evolved at least twice, in unrelated telomeric proteins Rif2 and Taz1. We identify the iDDR motif of human shelterin protein TRF2 as a third example of convergent evolution for this telomeric mechanism for binding MRN, despite the iDDR lacking sequence homology to the MIN motif. CtIP is required for activation of MRE11 nuclease action, and we provide evidence for binding of a short C-terminal region of CtIP to a RAD50 interface that partly overlaps with the iDDR binding site, indicating that the interaction is mutually exclusive. In addition, we show that the iDDR impairs the DNA binding activity of RAD50. These results highlight direct inhibition of MRN action as a crucial role of telomeric proteins across organisms and point to multiple mechanisms enforced by the iDDR to disable the many activities of the MRN complex.
Asunto(s)
Ácido Anhídrido Hidrolasas , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Unión Proteica , Proteínas de Unión a Telómeros , Telómero , Proteína 2 de Unión a Repeticiones Teloméricas , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Telómero/metabolismo , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ácido Anhídrido Hidrolasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Evolución Molecular , Roturas del ADN de Doble Cadena , Secuencias de Aminoácidos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sitios de Unión , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genéticaRESUMEN
The upregulation of RecQ helicases has been associated with cancer cell survival and resistance to chemotherapy, making them appealing targets for therapeutic intervention. In this study, twenty-nine novel quinazolinone derivatives were designed and synthesized. The anti-proliferative activity of all compounds was evaluated against 60 cancer cell lines at the National Cancer Institute Developmental Therapeutic Program, with six compounds (11f, 11g, 11k, 11n, 11p, and 11q) being promoted to a five-dose screen. Compound 11g demonstrated high cytotoxic activity against all examined cell lines. The compounds were further assayed for Bloom syndrome (BLM) helicase inhibition, where 11g, 11q, and 11u showed moderate activity. These compounds were counter-screened against WRN and RECQ1 helicases, where 11g moderately inhibited both enzymes. An ATP competition assay confirmed that the compounds bound to the ATP site of RecQ helicases, and molecular docking simulations were used to study the binding mode within the active site of BLM, WRN, and RECQ1 helicases. Compound 11g induced apoptosis in both HCT-116 and MDA-MB-231 cell lines, but also caused an G2/M phase cell cycle arrest in HCT-116 cells. This data revealed the potential of 11g as a modulator of cell cycle dynamics and supports its interaction with RecQ helicases. In addition, compound 11g displayed non-significant toxicity against FCH normal colon cells at doses up to 100 µM, which confirming its high safety margin and selectivity on cancer cells. Overall, these findings suggest compound 11g as a potential pan RecQ helicase inhibitor with high anticancer potency and a favorable safety margin and selectivity.
Asunto(s)
Antineoplásicos , RecQ Helicasas , Simulación del Acoplamiento Molecular , RecQ Helicasas/metabolismo , Quinazolinonas/farmacología , Antineoplásicos/farmacología , Adenosina TrifosfatoRESUMEN
The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.
Asunto(s)
Proteínas de Ciclo Celular , ADN de Cadena Simple , Proteínas de Ciclo Celular/metabolismo , ADN/química , Daño del ADN , ADN de Cadena Simple/genética , Humanos , Proteína de Replicación C/metabolismoRESUMEN
The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells - providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the 'W-loop' of Smc4 or 'F-loop' of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Microscopía por Crioelectrón , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismoRESUMEN
The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase activity, binding to the head-end of the complex to suppress turnover, irrespective of the DNA-bound status of the complex.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestructura , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/ultraestructura , ADN/metabolismo , Escherichia coli/metabolismo , Microscopía Electrónica de Transmisión , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , Fosforilación/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transglutaminasas , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Estructura Terciaria de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transglutaminasas/química , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
The extreme length of chromosomal DNA requires organizing mechanisms to both promote functional genetic interactions and ensure faithful chromosome segregation when cells divide. Microscopy and genome-wide contact frequency analyses indicate that intra-chromosomal looping of DNA is a primary pathway of chromosomal organization during all stages of the cell cycle. DNA loop extrusion has emerged as a unifying model for how chromosome loops are formed in cis in different genomic contexts and cell cycle stages. The highly conserved family of SMC complexes have been found to be required for DNA cis-looping and have been suggested to be the enzymatic core of loop extruding machines. Here, the current body of evidence available for the in vivo and in vitro action of SMC complexes is discussed and compared to the predictions made by the loop extrusion model. How SMC complexes may differentially act on chromatin to generate DNA loops and how they could work to generate the dynamic and functionally appropriate organization of DNA in cells is explored.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Animales , Archaea/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/ultraestructura , ADN/metabolismo , Eucariontes/metabolismo , HumanosRESUMEN
Mammalian polynucleotide kinase 3' phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3'-phosphates from, or by phosphorylating 5'-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3'-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5'-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites.
Asunto(s)
Enzimas Reparadoras del ADN/química , ADN de Cadena Simple/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Animales , Sitios de Unión , Cristalografía , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple/metabolismo , Ratones , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.
Asunto(s)
Empalme Alternativo , Complejo Multienzimático de Ribonucleasas del Exosoma/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas/enzimología , Proteínas de Neoplasias/biosíntesis , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Células HEK293 , Células HeLa , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas de Neoplasias/genética , Células THP-1RESUMEN
Patients with biallelic truncating mutations in PALB2 have a severe form of Fanconi anaemia (FA-N), with a predisposition for developing embryonal-type tumours in infancy. Here we describe two unusual patients from a single family, carrying biallelic PALB2 mutations, one truncating, c.1676_1677delAAinsG;(p.Gln559ArgfsTer2), and the second, c.2586+1G>A; p.Thr839_Lys862del resulting in an in frame skip of exon 6 (24 amino acids). Strikingly, the affected individuals did not exhibit the severe developmental defects typical of FA-N patients and initially presented with B cell non-Hodgkin lymphoma. The expressed p.Thr839_Lys862del mutant PALB2 protein retained the ability to interact with BRCA2, previously unreported in FA-N patients. There was also a large increased chromosomal radiosensitivity following irradiation in G2 and increased sensitivity to mitomycin C. Although patient cells were unable to form Rad51 foci following exposure to either DNA damaging agent, U2OS cells, in which the mutant PALB2 with in frame skip of exon 6 was induced, did show recruitment of Rad51 to foci following damage. We conclude that a very mild form of FA-N exists arising from a hypomorphic PALB2 allele.
Asunto(s)
Anemia de Fanconi/genética , Linfoma no Hodgkin/genética , Proteínas Nucleares/genética , Recombinasa Rad51/genética , Proteínas Supresoras de Tumor/genética , Alelos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Cromosomas/genética , Daño del ADN/genética , Anemia de Fanconi/patología , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Linfoma no Hodgkin/patología , MutaciónRESUMEN
SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Replicación del ADN , Humanos , Datos de Secuencia Molecular , Unión Proteica , Recombinación Genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.
Asunto(s)
Antígenos Nucleares/metabolismo , ADN Ligasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos Nucleares/genética , Línea Celular , Supervivencia Celular , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Autoantígeno Ku , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Rayos UltravioletaRESUMEN
Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5'-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a 'humanized' form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.
Asunto(s)
Hidrolasas Diéster Fosfóricas/metabolismo , Riboflavina/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Hidrolasas Diéster Fosfóricas/química , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Riboflavina/análogos & derivados , Riboflavina/farmacología , TemperaturaRESUMEN
Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Animales , Sitios de Unión , Células CHO , Línea Celular Tumoral , Supervivencia Celular , Cricetulus , Daño del ADN , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estructura Terciaria de Proteína , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas/genética , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Redes Reguladoras de Genes/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Activating somatic PIK3CA mutations underlie a growing heterogeneous spectrum of segmental overgrowth disorders. We report the identification and evaluation of a novel de novo constitutional PIK3CA mutation (NM_006218.2:c.335T>A, p.Ile112Asn) in a child with congenital megalencephaly and macrosomia. Functional characterization of patient cells using a variety of endpoints demonstrates increased phosphatidylinositol-3-kinase (PI3K) activity. The mutation lies in a linker region adjacent to the p85 (PIK3R2) binding domain of the p110α (PIK3CA) catalytic subunit of PI3K. We show that altered stoichiometry within the p85-p110 complex likely underlies the hyperactive PI3K-AKT-mTOR signaling in this instance. Our findings expand upon the recently proposed "PIK3CA-related overgrowth spectrum" associated with PIKC3A mutations and PI3K hyperactivation, adding constitutional PIK3CA mutations as an underlying cause of megalencephaly and macrosomia in newborns.
Asunto(s)
Fosfatidilinositol 3-Quinasas/genética , Niño , Fosfatidilinositol 3-Quinasa Clase I , Humanos , Masculino , Megalencefalia/genética , MutaciónRESUMEN
Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Ribosómico , Silenciador del Gen , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.