Protocol for the quantitative identification of endogenously ISGylated proteins from mammalian cell lines.

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.

ISG15: A link between innate immune signaling, DNA replication, and genome stability.

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.

ISG15 conjugation to proteins on nascent DNA mitigates DNA replication stress.

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.

TopBP1: A BRCT-scaffold protein functioning in multiple cellular pathways.

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.

Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4(TopBP1).

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.

The Rad4(TopBP1) ATR-activation domain functions in G1/S phase in a chromatin-dependent manner.

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).