RESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Análisis de Secuencia de ADN/métodos , Reparación del ADNRESUMEN
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
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.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa II/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Replicación ViralRESUMEN
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
Arrested replication forks, when restarted by homologous recombination, result in error-prone DNA syntheses and non-allelic homologous recombination. Fission yeast RTS1 is a model fork barrier used to probe mechanisms of recombination-dependent restart. RTS1 barrier activity is entirely dependent on the DNA binding protein Rtf1 and partially dependent on a second protein, Rtf2. Human RTF2 was recently implicated in fork restart, leading us to examine fission yeast Rtf2's role in more detail. In agreement with previous studies, we observe reduced barrier activity upon rtf2 deletion. However, we identified Rtf2 to be physically associated with mRNA processing and splicing factors and rtf2 deletion to cause increased intron retention. One of the most affected introns resided in the rtf1 transcript. Using an intronless rtf1, we observed no reduction in RFB activity in the absence of Rtf2. Thus, Rtf2 is essential for correct rtf1 splicing to allow optimal RTS1 barrier activity.
Asunto(s)
Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Empalme del ARN , Procesamiento Postranscripcional del ARN , Intrones , Replicación del ADN/genéticaRESUMEN
Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.
Asunto(s)
Proteínas de Ciclo Celular , Microcefalia , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microcefalia/genética , Reparación del ADN/genética , Cromosomas/metabolismo , Inestabilidad Genómica , Proteínas de Unión al ADN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cromosómicas no Histona/metabolismoRESUMEN
Detection of cytosolic DNA is a central element of the innate immunity system against viral infection. The Ku heterodimer, a component of the NHEJ pathway of DNA repair in the nucleus, functions as DNA sensor that detects dsDNA of viruses that replicate in the cytoplasm. Vaccinia virus expresses two proteins, C4 and C16, that inactivate DNA sensing and enhance virulence. The structural basis for this is unknown. Here we determine the structure of the C16 - Ku complex using cryoEM. Ku binds dsDNA by a preformed ring but C16 sterically blocks this access route, abrogating binding to a dsDNA end and its insertion into DNA-PK, thereby averting signalling into the downstream innate immunity system. C4 replicates these activities using a domain with 54% identity to C16. Our results reveal how vaccinia virus subverts the capacity of Ku to recognize viral DNA.
Asunto(s)
Proteínas de Unión al ADN , Virus Vaccinia , Virus Vaccinia/genética , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , ADN/metabolismo , Proteína Quinasa Activada por ADN/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 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 cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 - a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , FosforilaciónRESUMEN
The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.
Asunto(s)
Secuencias de Aminoácidos , ADN Helicasas/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ADN Helicasas/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Inestabilidad Genómica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/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 essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Imagen Individual de MoléculaRESUMEN
CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN, a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Animales , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Células Sf9RESUMEN
BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response; however, selective small molecule inhibitors of defined mechanism are currently lacking. Here, we identify and characterise a specific inhibitor of BLM's ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.
Asunto(s)
RecQ Helicasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , ADN/metabolismo , ADN Cruciforme , ADN de Cadena Simple , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli , Ensayos Analíticos de Alto Rendimiento , Humanos , RecQ Helicasas/metabolismoRESUMEN
The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Sustancias Macromoleculares/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Unión Proteica/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Coordination of the cellular response to DNA damage is organised by multi-domain 'scaffold' proteins, including 53BP1 and TOPBP1, which recognise post-translational modifications such as phosphorylation, methylation and ubiquitylation on other proteins, and are themselves carriers of such regulatory signals. Here we show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 and TOPBP1. BRCT domains of TOPBP1 selectively bind conserved phosphorylation sites in the N-terminus of 53BP1. Mutation of these sites does not affect formation of 53BP1 or ATM foci following DNA damage, but abolishes recruitment of TOPBP1, ATR and CHK1 to 53BP1 damage foci, abrogating cell cycle arrest and permitting progression into S-phase. TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-RAD1-HUS1, allowing these damage recognition factors to bind simultaneously to the same TOPBP1 molecule and cooperate in ATR activation in the G1 DNA damage checkpoint.
Asunto(s)
Proteínas Portadoras/química , Daño del ADN/genética , Proteínas de Unión al ADN/química , Complejos Multiproteicos/química , Proteínas Nucleares/química , Proteína 1 de Unión al Supresor Tumoral P53/química , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Metilación , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Fosforilación , Unión Proteica/genética , Conformación Proteica , Dominios Proteicos/genética , Procesamiento Proteico-Postraduccional/genética , Fase S/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitinación/genéticaRESUMEN
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
XRCC1 accelerates repair of DNA single-strand breaks by acting as a scaffold protein for the recruitment of Polß, LigIIIα, and end-processing factors, such as PNKP and APTX. XRCC1 itself is recruited to DNA damage through interaction of its central BRCT domain with poly(ADP-ribose) chains generated by PARP1 or PARP2. XRCC1 is believed to interact directly with DNA at sites of damage, but the molecular basis for this interaction within XRCC1 remains unclear. We now show that the central BRCT domain simultaneously mediates interaction of XRCC1 with poly(ADP-ribose) and DNA, through separate and non-overlapping binding sites on opposite faces of the domain. Mutation of residues within the DNA binding site, which includes the site of a common disease-associated human polymorphism, affects DNA binding of this XRCC1 domain in vitro and impairs XRCC1 recruitment and retention at DNA damage and repair of single-strand breaks in vivo.
Asunto(s)
Roturas del ADN de Cadena Simple , Poli Adenosina Difosfato Ribosa/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Humanos , Poli Adenosina Difosfato Ribosa/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
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.