RESUMEN
Efficient double-strand break repair in eukaryotes requires manipulation of chromatin structure. ATP-dependent chromatin remodelling enzymes facilitate different DNA repair pathways, during different stages of the cell cycle and in varied chromatin environments. The contribution of remodelling factors to double-strand break repair within heterochromatin during G2 is unclear. The human HELLS protein is a Snf2-like chromatin remodeller family member and is mutated or misregulated in several cancers and some cases of ICF syndrome. HELLS has been implicated in the DNA damage response, but its mechanistic function in repair is not well understood. We discover that HELLS facilitates homologous recombination at two-ended breaks and contributes to repair within heterochromatic regions during G2. HELLS promotes initiation of HR by facilitating end-resection and accumulation of CtIP at IR-induced foci. We identify an interaction between HELLS and CtIP and establish that the ATPase domain of HELLS is required to promote DSB repair. This function of HELLS in maintenance of genome stability is likely to contribute to its role in cancer biology and demonstrates that different chromatin remodelling activities are required for efficient repair in specific genomic contexts.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Roturas del ADN de Doble Cadena , ADN Helicasas/genética , Recombinación Homóloga/genética , Daño del ADN/genética , Reparación del ADN/genética , Genoma Humano/genética , Inestabilidad Genómica/genética , Heterocromatina/genética , HumanosRESUMEN
General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.
Asunto(s)
Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Humanos , Espectrometría de Masas , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Factor de Transcripción TFIID/químicaRESUMEN
Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein-DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein-DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Inestabilidad Cromosómica , Replicación del ADN , Proteínas de Unión al ADN/fisiología , ADN/química , Proteínas de Saccharomyces cerevisiae/fisiología , Ciclo Celular , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Hongos/química , Eliminación de Gen , Genotipo , Fosforilación , Plásmidos/metabolismo , Saccharomycetales/genética , Procesos EstocásticosRESUMEN
Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Roturas del ADN , Reparación del ADN , Regulación de la Expresión Génica , Factores de Transcripción/fisiología , Sitios de Unión , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Factores de Transcripción/química , Factores de Transcripción/metabolismo , UbiquitinaciónRESUMEN
BAF180, a subunit of the PBAF chromatin remodeling complex, is frequently mutated in cancer. Although PBAF regulates transcription, it remains unclear whether this is what drives tumorigenesis in cells lacking BAF180. Based on data from yeast, we hypothesized that BAF180 may prevent tumorigenesis by promoting cohesion. Here, we show BAF180 is required for centromeric cohesion in mouse and human cells. Mutations identified in tumor samples are unable to support this activity, and also compromise cohesion-dependent functions in yeast. We provide evidence of genome instability in line with loss of cohesion, and importantly, we find dynamic chromosome instability following DNA damage in cells lacking BAF180. These data demonstrate a function for BAF180 in promoting genome stability that is distinct from its well-characterized role in transcriptional regulation, uncovering a potent mechanism for its tumor-suppressor activity.
Asunto(s)
Aneuploidia , Inestabilidad Genómica , Proteínas HMGB/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Humanos , RatonesRESUMEN
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
The INO80 chromatin remodeling complex functions in transcriptional regulation, DNA repair, and replication. Here we uncover a novel role for INO80 in regulating chromosome segregation. First, we show that the conserved Ies6 subunit is critical for INO80 function in vivo. Strikingly, we found that loss of either Ies6 or the Ino80 catalytic subunit results in rapid increase in ploidy. One route to polyploidy is through chromosome missegregation due to aberrant centromere structure, and we found that loss of either Ies6 or Ino80 leads to defective chromosome segregation. Importantly, we show that chromatin structure flanking centromeres is altered in cells lacking these subunits and that these alterations occur not in the Cse4-containing centromeric nucleosome, but in pericentric chromatin. We provide evidence that these effects are mediated through misincorporation of H2A.Z, and these findings indicate that H2A.Z-containing pericentric chromatin, as in higher eukaryotes with regional centromeres, is important for centromere function in budding yeast. These data reveal an important additional mechanism by which INO80 maintains genome stability.
Asunto(s)
Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Poliploidía , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrómero/química , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Daño del ADN , Regulación Fúngica de la Expresión Génica , Histonas/genética , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Complejos Multiproteicos/metabolismo , Animales , ADN Helicasas/metabolismo , Humanos , Modelos BiológicosRESUMEN
In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ciclo Celular , Línea Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Eliminación de Gen , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/fisiología , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Chromatin remodelling complexes alter the structure of chromatin and have central roles in all DNA-templated activities, including regulation of gene expression and DNA repair. Mutations in subunits of the PBAF (polybromo/Brg1-associated factor) or SWI/SNF-B remodelling complex, including BAF180, are frequently associated with cancer. There are six potential acetyl-lysine-binding BDs (bromodomains) in BAF180, which may function to target the PBAF complex to promoters or sites of DNA repair. In the present review, we discuss what is currently known about the BDs of BAF180 and their potential significance in cancer.
Asunto(s)
Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Humanos , Mutación/genética , Neoplasias/genética , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismoRESUMEN
The RSC chromatin remodeling complex has been implicated in contributing to DNA double-strand break (DSB) repair in a number of studies. Both survival and levels of H2A phosphorylation in response to damage are reduced in the absence of RSC. Importantly, there is evidence for two isoforms of this complex, defined by the presence of either Rsc1 or Rsc2. Here, we investigated whether the two isoforms of RSC provide distinct contributions to DNA damage responses. First, we established that the two isoforms of RSC differ in the presence of Rsc1 or Rsc2 but otherwise have the same subunit composition. We found that both rsc1 and rsc2 mutant strains have intact DNA damage-induced checkpoint activity and transcriptional induction. In addition, both strains show reduced non-homologous end joining activity and have a similar spectrum of DSB repair junctions, suggesting perhaps that the two complexes provide the same functions. However, the hypersensitivity of a rsc1 strain cannot be complemented with an extra copy of RSC2, and likewise, the hypersensitivity of the rsc2 strain remains unchanged when an additional copy of RSC1 is present, indicating that the two proteins are unable to functionally compensate for one another in DNA damage responses. Rsc1, but not Rsc2, is required for nucleosome sliding flanking a DNA DSB. Interestingly, while swapping the domains from Rsc1 into the Rsc2 protein does not compromise hypersensitivity to DNA damage suggesting they are functionally interchangeable, the BAH domain from Rsc1 confers upon Rsc2 the ability to remodel chromatin at a DNA break. These data demonstrate that, despite the similarity between Rsc1 and Rsc2, the two different isoforms of RSC provide distinct functions in DNA damage responses, and that at least part of the functional specificity is dictated by the BAH domains.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/fisiología , Reparación del ADN , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Daño del ADN , Proteínas Fúngicas , Isoformas de Proteínas , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genéticaRESUMEN
DNA double strand breaks (DSBs) are potentially serious chromosomal lesions. However, cells sometimes deliberately cleave their own DNA to facilitate certain chromosomal processes, and there is much interest in how such self-inflicted breaks are effectively managed. Eukaryotic DSBs occur in the context of chromatin and the RSC chromatin-remodeling ATPase complex has been shown to promote DSB repair at the budding yeast MAT locus DSB, created by the HO endonuclease during mating type switching. We show that the role of RSC at MAT is highly specialized. The Rsc1p subunit of RSC directs nucleosome sliding immediately after DSB creation at both MAT and generally and is required for efficient DNA damage-induced histone H2A phosphorylation and strand resection during repair by homologous recombination. However, the Rsc2p and Rsc7p subunits are additionally required to set up a basal MAT locus structure. This RSC-dependent chromatin structure at MAT ensures accessibility to the HO endonuclease. The RSC complex therefore has chromatin remodeling roles both before and after DSB induction at MAT, promoting both DNA cleavage and subsequent repair.
Asunto(s)
Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Regulación Fúngica de la Expresión Génica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Fúngicas , Histonas/química , Modelos Biológicos , Modelos Genéticos , Nucleosomas/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Recombinación Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.