Dissection of DNA Damage Responses Using Multiconditional Genetic Interaction Maps

Guénolé, Aude; Srivas, Rohith; Vreeken, Kees; Wang, Ze Zhong; Wang, Shuyi; Krogan, Nevan J.; Ideker, Trey; Attikum, Haico van

doi:10.1016/j.molcel.2012.11.023

Cited by 95 publications

(122 citation statements)

References 47 publications

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“…Additionally, there were nonessential components of the APC and cyclins that were tested and failed to show a reduction in NHEJ efficiency, suggesting that only certain components of the APC and cyclins tend to function specifically in NHEJ by cooperating with Bub, while others may have either a role in an unrelated process or even an opposing (i.e., antagonistic) function with Bub. This is indeed supported by the findings from a recent bub1 and bub2 genetic screen under DNA-damaging conditions (55), which showed positively and negatively correlated genetic interaction profiles with components of the APC.…”

Section: Discussionsupporting

confidence: 66%

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Jessulat

Malty

Nguyen-Tran

et al. 2015

Molecular and Cellular Biology

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The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmidbased NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.T he repair of DNA double-strand breaks (DSBs) is an essential process required for the preservation of genome integrity and the normal functioning of the cell (1). These cytotoxic lesions are repaired by major DSB repair pathways, including the homologous recombination (HR) (2) and nonhomologous end-joining (NHEJ) systems (1). While the former is the prevalent pathway in the unicellular budding yeast Saccharomyces cerevisiae (3), the latter is more prevalent in mammalian cells, especially those that are quiescent (4), and can repair DNA lesions even if there is no homologous strand (5). Notably, the impairment of NHEJ in mammalian cells is frequently linked to genomic instability, cancer, and lymphoid V(D)J (i.e., variable, diversity, and joining gene segments) recombination defects. Therefore, a detailed molecular understanding of this pathway would provide critical insight into the genetic risk factors related to carcinogenesis or immunological disorders (6).As in mammalian cells, the core components of the classical NHEJ pathway in S. cerevisiae depends on three major complexes, YKu (Ku), MRX, and DNL4, which are rapidly recruited to DSBs (7). Initially, the yeast Ku heterodimer (Ku70/80) binds to each end of a DSB, serving as an anchor for protein complexes involved in securing and annealing the break, also suppressing the competing HR pathway (8). After this, the DSB processing complex MRX (Mre11-Rad50-Xrs2), which acts at an early stage of both the NHEJ and HR repair pathways (9), spans the lesions so that the DNA ligase complex, DNL4 (i.e., Dnl4-Lif1-Nej1), can rejoin the DSB ends (10).While the actions of these core protein complexes in y...

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Section: Discussionsupporting

confidence: 66%

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Jessulat

Malty

Nguyen-Tran

et al. 2015

Molecular and Cellular Biology

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“…The EMAP analysis was performed as described in Guenole et al (2013), and suppressive (not synergistic) effects were elucidated in Hustedt et al (2015).…”

Section: Emapmentioning

confidence: 99%

Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

et al. 2016

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Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1-Ddc2 (ATR-ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription-replication fork collision.

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“…Through binding to its cellular target FKBP12, it inhibits the activity of the phosphatase calcineurin, which results in inhibition of T-cell activation and IL-2 production (35). Simvastatin is a member of the statin class of cholesterol-lowering drugs that act through binding to and inhibiting the activity of HMGCR, the rate-limiting enzyme in the cholesterol biosynthetic pathway (36). FK506 and simvastatin were coupled to MTX through a hexaethyleneglycol linker that served to reduce potential steric hindrance of binding to eDHFR and the drug target at either end of the chemical dimerizer (21) (Fig.…”

Section: Kiss Three-hybrid Analysis Of Interactions Between Small Molmentioning

confidence: 99%

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Lievens

Gerlo

Lemmens

et al. 2014

Molecular & Cellular Proteomics

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Probably every cellular process is governed by proteinprotein interaction (PPIs), which are often highly dynamic in nature being modulated by in-or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent ␤-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken A protein's function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI) 1 maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra-and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow

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Dissection of DNA Damage Responses Using Multiconditional Genetic Interaction Maps

Cited by 95 publications

References 47 publications

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

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