RESUMO
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.
Assuntos
Proteínas de Transporte/genética , Instabilidade Cromossômica/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Proteínas Nucleares/genética , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Fase G1/genética , Genoma Humano/genética , Instabilidade Genômica/genética , Histonas , Humanos , Fosforilação , Transdução de Sinais/genéticaRESUMO
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.
Assuntos
Hidrolases Anidrido Ácido , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , Ligação Proteica , Proteínas de Ligação a Telômeros , Telômero , Proteína 2 de Ligação a Repetições Teloméricas , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/genética , Telômero/metabolismo , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Hidrolases Anidrido Ácido/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Evolução Molecular , Quebras de DNA de Cadeia Dupla , Motivos de Aminoácidos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sítios de Ligação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
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.
Assuntos
Antineoplásicos , RecQ Helicases , Simulação de Acoplamento Molecular , RecQ Helicases/metabolismo , Quinazolinonas/farmacologia , Antineoplásicos/farmacologia , Trifosfato de AdenosinaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , DNA de Cadeia Simples , Proteínas de Ciclo Celular/metabolismo , DNA/química , Dano ao DNA , DNA de Cadeia Simples/genética , Humanos , Proteína de Replicação C/metabolismoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/metabolismo , Escherichia coli/metabolismo , Microscopia Eletrônica de Transmissão , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA , Proteínas Nucleares/metabolismo , Fosforilação/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transglutaminases , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transglutaminases/química , Transglutaminases/genética , Transglutaminases/metabolismoRESUMO
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.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Animais , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/ultraestrutura , DNA/metabolismo , Eucariotos/metabolismo , HumanosRESUMO
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.
Assuntos
Enzimas Reparadoras do DNA/química , DNA de Cadeia Simples/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Animais , Sítios de Ligação , Cristalografia , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Camundongos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Processamento Alternativo , Complexo Multienzimático de Ribonucleases do Exossomo/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/enzimologia , Proteínas de Neoplasias/biossíntese , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HEK293 , Células HeLa , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Neoplasias/genética , Células THP-1RESUMO
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.
Assuntos
Anemia de Fanconi/genética , Linfoma não Hodgkin/genética , Proteínas Nucleares/genética , Rad51 Recombinase/genética , Proteínas Supressoras de Tumor/genética , Alelos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Cromossomos/genética , Dano ao DNA/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação N da Anemia de Fanconi , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Linfoma não Hodgkin/patologia , MutaçãoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Replicação do DNA , Humanos , Dados de Sequência Molecular , Ligação Proteica , Recombinação Genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Riboflavina/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Diester Fosfórico Hidrolases/química , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Riboflavina/análogos & derivados , Riboflavina/farmacologia , TemperaturaRESUMO
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.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular , Cricetulus , Dano ao DNA , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Estrutura Terciária de Proteína , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
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.
Assuntos
Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas/genética , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Redes Reguladoras de Genes/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
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.
Assuntos
Proteínas Cromossômicas não Histona/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico , Inativação Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Espectrometria de Fluorescência/métodos , Sequência de Bases , Análise Custo-Benefício , Avaliação Pré-Clínica de Medicamentos/economia , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Mutação , Oligonucleotídeos/genética , Diester Fosfórico Hidrolases/genética , Espectrometria de Fluorescência/economiaRESUMO
During meiosis, Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronized Saccharomyces cerevisiae meiotic cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases , Meiose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Análise de Sequência de DNA/métodos , Reparo do DNARESUMO
Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.