The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.

Cells respond to DNA double-strand breaks (DSBs) by recruiting factors such as the DNAdamage mediator protein MDC1, p53-binding protein 1 (53BP1) and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 via phospho-dependent interactions between the RNF8 forkhead-associated (FHA) domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ATM. We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage suggesting that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.DNA DSBs are highly cytotoxic lesions; and to ensure that they are repaired with minimal impact on genome stability, cells mount a complex DNA-damage response (DDR) that includes the spatial reorganization of DSB repair and signaling proteins into subnuclear structures -ionizing radiation-induced foci (IRIF) -that surround DSB sites (1, 2). Most IRIF formation depends on phosphorylation of the histone variant H2AX (to form γH2AX) by the DNA-PK and ATM protein kinases (3-6). The γH2AX epitope is bound by MDC1 (7-10) that then promotes IRIF formation by other proteins, including 53BP1, Nijmegenbreakage-syndrome protein NBS1 and BRCA1 (11,12). BRCA1 recruitment to IRIF requires its interaction with the ubiquitin-binding protein RAP80 (13)(14)(15)(16) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts identify RNF8 as the prime ubiquitin ligase for poly-ubiquitination at DSB sites, define its functional importance in the DDR, and establish how RNF8 is recruited to sites of DNA damage via interactions with MDC1.MDC1 is phosphorylated in an ATM-dependent manner in response to ionizing radiation (IR) (11,12). Potential ATM target sites (consensus S/T-Q) cluster in the MDC1 Nterminus, the most notable being four adjacent motifs conforming to the consensus TQXF (Figs. 1A and S1). Significantly, antibodies raised against a peptide encoding phospho-T719 ( Fig. S2A) indicated that it is targeted by ATM in vitro (Fig. 1B), and in vivo ( Fig. 1C and S2B). However, in vitro assays with bacterially-expressed MDC1 fragments revealed that T719 was not the only site of ATM modification. Phosphorylation was only abolished when an "AQXF" mutant protein bearing threonine-to-alanine substitutions in all four TQXF motifs was used as substrate (Fig. 1D). These data and the recent identification of another TQXF site (T752) as an ATM target (17) therefore imply that MDC1 TQXF motifs are likely all modified by ATM and may function redundantly with one another.To address the function of the MDC1 TQXF motifs, we used siRNA to deplete endogenous MDC1 i...

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Ubiquitylation and sumoylation, the covalent attachment of the polypeptides ubiquitin and SUMO, respectively, to target proteins, are pervasive mechanisms for controlling cellular functions. Here, we summarize the key steps and enzymes involved in ubiquitin and SUMO conjugation and provide an overview of how they are crucial for maintaining genome stability. Specifically, we review research that has revealed how ubiquitylation and sumoylation regulate and coordinate various pathways of DNA damage recognition, signaling, and repair at the biochemical, cellular, and whole-organism levels. In addition to providing key insights into the control and importance of DNA repair and associated processes, such work has established paradigms for regulatory control that are likely to extend to other cellular processes and that may provide opportunities for better understanding and treatment of human disease.

The correct duplication and transmission of genetic material to daughter cells is the primary objective of the cell division cycle. DNA replication and chromosome segregation present both challenges and opportunities for DNA repair pathways that safeguard genetic information. As a consequence, there is a profound, two-way connection between DNA repair and cell cycle control. Here, we review how DNA repair processes, and DNA double-strand break repair in particular, are regulated during the cell cycle to optimize genomic integrity.

The tissue‐restricted GATA‐4 transcription factor and Nkx2‐5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA‐4 or Nkx2‐5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA‐4 and Nkx2‐5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter—the only known target for Nkx2‐5. Co‐expression of GATA‐4 and Nkx2‐5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2‐5–GATA‐4 interaction, seen in vitro and in vivo, which maps to the C‐terminal zinc finger of GATA‐4 and a C‐terminus extension; similarly, a C‐terminally extended homeodomain of Nkx2‐5 is required for GATA‐4 binding. Structure/function studies suggest that binding of GATA‐4 to the C‐terminus autorepressive domain of Nkx2‐5 may induce a conformational change that unmasks Nkx2‐5 activation domains. GATA‐6 cannot substitute for GATA‐4 for interaction with Nkx2‐5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co‐expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.