RESUMEN
Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.
Asunto(s)
Aminoaciltransferasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , ARN de Transferencia de Leucina/genética , Anticodón , Cinética , Conformación de Ácido Nucleico , Nucleótidos , Aminoacil-ARN de Transferencia , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/metabolismo , Especificidad por Sustrato , Aminoacilación de ARN de TransferenciaRESUMEN
Activation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response.
Asunto(s)
Proteínas Portadoras/química , Momento de Replicación del ADN , Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Transactivadores/química , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Secuencias Repetitivas de AminoácidoRESUMEN
BRCA1 C-terminal (BRCT) domains are integral signaling modules in the DNA damage response (DDR). Aside from their established roles as phospho-peptide binding modules, BRCT domains have been implicated in phosphorylation-independent protein interactions, DNA binding and poly(ADP-ribose) (PAR) binding. These numerous functions can be attributed to the diversity in BRCT domain structure and architecture, where domains can exist as isolated single domains or assemble into higher order homo- or hetero- domain complexes. In this review, we incorporate recent structural and biochemical studies to demonstrate how structural features allow single and tandem BRCT domains to attain a high degree of functional diversity.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , ADN/química , ADN/metabolismo , Reparación del ADN , Fosforilación , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Daño del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes de Cambio , Genes cdc , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Treonina/genéticaRESUMEN
The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , ADN Helicasas/química , ADN Helicasas/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Portadoras/genética , Cristalografía por Rayos X , ADN/biosíntesis , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Mutagénesis , Proteínas Nucleares/genética , Fosforilación , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Human TopBP1 plays a critical role in the control of DNA replication checkpoint. In this study, we report a specific interaction between TopBP1 and BACH1/FANCJ, a DNA helicase involved in the repair of DNA crosslinks. The TopBP1/BACH1 interaction is mediated by the very C-terminal tandem BRCT domains of TopBP1 and S phase-specific phosphorylation of BACH1 at Thr 1133 site. Interestingly, we demonstrate that depletion of TopBP1 or BACH1 attenuates the loading of RPA on chromatin. Moreover, both TopBP1 and BACH1 are required for ATR-dependent phosphorylation events in response to replication stress. Taken together, our data suggest that BACH1 has an unexpected early role in replication checkpoint control. A specific interaction between TopBP1 and BACH1 is likely to be required for the extension of single-stranded DNA regions and RPA loading following replication stress, which is a prerequisite for the subsequent activation of replication checkpoint.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Proteínas Portadoras/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/fisiología , Proteínas Nucleares/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/metabolismo , Daño del ADN , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Replicación A/metabolismoRESUMEN
Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.
Asunto(s)
Proteínas Portadoras/química , Cristalografía por Rayos X/métodos , Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Secuencia de Consenso , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Alineación de Secuencia , Secuencias Repetidas en TándemRESUMEN
To maintain genome stability, cells respond to DNA damage by activating signalling pathways that govern cell-cycle checkpoints and initiate DNA repair. Cell-cycle checkpoint controls should connect with DNA repair processes, however, exactly how such coordination occurs in vivo is largely unknown. Here we describe a new role for the E3 ligase RAD18 as the integral component in translating the damage response signal to orchestrate homologous recombination repair (HRR). We show that RAD18 promotes homologous recombination in a manner strictly dependent on its ability to be recruited to sites of DNA breaks and that this recruitment relies on a well-defined DNA damage signalling pathway mediated by another E3 ligase, RNF8. We further demonstrate that RAD18 functions as an adaptor to facilitate homologous recombination through direct interaction with the recombinase RAD51C. Together, our data uncovers RAD18 as a key factor that orchestrates HRR through surveillance of the DNA damage signal.