RESUMO
Ubiquitin-like protein ISG15 plays an important role in an array of cellular functions via its covalent attachment to target proteins (ISGylation). Here, we present a protocol for the identification of ISGylated proteins that avoids the caveats associated with ISG15 overexpression and minimizes the likelihood of false positives. We describe steps for the tagging of endogenous ISG15, followed by genotyping and clone selection. We then detail steps for ISGylation induction, the isolation of ISGylated proteins, and their identification via quantitative mass spectrometry. For complete details on the use and execution of this protocol, please refer to Wardlaw and Petrini.1.
Assuntos
Citocinas , Ubiquitinas , Animais , Citocinas/genética , Citocinas/metabolismo , Ubiquitinas/genética , Ubiquitinas/química , Ubiquitinas/metabolismo , Linhagem Celular , Mamíferos/metabolismoRESUMO
Interferon stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is highly induced upon activation of interferon signaling and cytoplasmic DNA sensing pathways. As part of the innate immune system ISG15 acts to inhibit viral replication and particle release via the covalent conjugation to both viral and host proteins. Unlike ubiquitin, unconjugated ISG15 also functions as an intracellular and extra-cellular signaling molecule to modulate the immune response. Several recent studies have shown ISG15 to also function in a diverse array of cellular processes and pathways outside of the innate immune response. This review explores the role of ISG15 in maintaining genome stability, particularly during DNA replication, and how this relates to cancer biology. It puts forth the hypothesis that ISG15, along with DNA sensors, function within a DNA replication fork surveillance pathway to help maintain genome stability.
Assuntos
Citocinas , Interferons , DNA , Replicação do DNA , Imunidade Inata , Ubiquitinas/genética , Ubiquitinas/metabolismo , Humanos , AnimaisRESUMO
The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among double strand break (DSB) repair proteins for its association with the DNA replication fork. Here we show that Mre11 complex inactivation causes DNA replication stress and changes in the abundance of proteins associated with nascent DNA. One of the most highly enriched proteins at the DNA replication fork upon Mre11 complex inactivation was the ubiquitin like protein ISG15. Mre11 complex deficiency and drug induced replication stress both led to the accumulation of cytoplasmic DNA and the subsequent activation of innate immune signaling via cGAS-STING-Tbk1. This led to ISG15 induction and protein ISGylation, including constituents of the replication fork. ISG15 plays a direct role in preventing replication stress. Deletion of ISG15 was associated with replication fork stalling, tonic ATR activation, genomic aberrations, and sensitivity to aphidicolin. These data reveal a previously unrecognized role for ISG15 in mitigating DNA replication stress and promoting genomic stability.
Assuntos
Reparo do DNA , Replicação do DNA , Afidicolina , DNA/genética , Dano ao DNA , Replicação do DNA/genética , Nucleotidiltransferases/genética , Ubiquitinas/genéticaRESUMO
Human TopBP1 contains nine BRCT domains and functions in DNA replication initiation, checkpoint signalling, DNA repair and influences transcriptional control. TopBP1 and its homologues have been the subject of numerous scientific publications since the last comprehensive review in 2005, emerging as a key scaffold protein that links crucial components within these distinct cellular processes. This review focuses on recently published work, with particular emphasis on structural insights into TopBP1 function and the binding partners identified for DNA replication initiation, DNA-dependent checkpoints, DNA repair and transcription. We further summarise what is known about TopBP1 and links to human disease.
Assuntos
Proteínas de Transporte/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Pontos de Checagem do Ciclo Celular , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrutura Terciária de ProteínaRESUMO
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
DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4(TopBP1) AAD-defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage-independent assay for checkpoint activation that is Rad4(TopBP1) AAD-dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb2(53BP1). Consistent with a model where Rad4(TopBP1) AAD-dependent checkpoint activation is ssDNA/RPA-independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4(TopBP1) AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4(TopBP1) AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD-dependent Rad3(ATR) checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein-chromatin interactions.