Phosphoproteomics Reveals Distinct Modes of Mec1/ATR Signaling during DNA Replication

Oliveira, Francisco Meirelles Bastos de; Kim, Dongsung; Cussiol, José Renato Rosa; Das, Jishnu; Jeong, Min Cheol; Doerfler, Lillian; Schmidt, Kristina; Yu, Haiyuan; Smolka, Marcus B.

doi:10.1016/j.molcel.2015.03.026

Cited by 40 publications

(71 citation statements)

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“…This is consistent with the fact that Mec1 modifies largely different sets of proteins in unperturbed S-phase cells than it does in S-phase cells under stress (Hoch et al 2013;Bastos de Oliveira et al 2015). Whereas HU-induced removal of RNAPII from chromatin requires Mec1, it does not require the downstream kinase Rad53/CHK2.…”

Section: Sites Of Replication Fork-transcription Collision Show Rnapisupporting

confidence: 84%

“…In addition, the ATPase subunit itself, Ino80, is phosphorylated at residue Ser115 by Rad53 ( Fig. 3A; Supplemental Table S3; Bastos de Oliveira et al 2015). We performed an additional phosphoproteomic analysis to compare modification states between wildtype and rad53Δsml1Δ cells and found that Arp8 in INO80C and Ctr9 in PAF1C are phosphorylated by Rad53 upon exposure to HU as well (Supplemental Table S4).…”

Section: The Ino80-mec1-paf1 Complexes Harbor Multiple Phosphorylatiomentioning

confidence: 98%

“…Recent phosphoproteomic analyses have shown that many factors involved in chromatin remodeling and transcription, besides DNA replication and repair factors, are targets of checkpoint kinases (Bastos de Oliveira et al 2015;Hustedt et al 2015). Among these is the INO80 complex (INO80C), a 15-subunit nucleosome remodeler conserved from yeast to humans that slides and modifies the composition of nucleosomes on a DNA template (Gerhold et al 2015).…”

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confidence: 99%

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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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Section: Sites Of Replication Fork-transcription Collision Show Rnapisupporting

confidence: 84%

Section: The Ino80-mec1-paf1 Complexes Harbor Multiple Phosphorylatiomentioning

confidence: 98%

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confidence: 99%

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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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“…These results suggest that numerous substrates may exist in other organisms as well. While proteomic studies in Saccharomyces cerevisiae have indeed supported this (18)(19)(20), we predicted that many additional substrates remained to be discovered. We profiled DDR phosphorylation in budding yeast with the hope that discovery of new substrates would uncover pathways that reveal critical connections between species.…”

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confidence: 90%

Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks

Zhou

Elia

Naylor

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through largescale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.enomes of all organisms constantly experience life-threatening chemical and structural alterations because of the highly reactive chemical environments in which they reside and endogenous errors that occur during genome replication (1, 2). The ability to repair these lesions and maintain genomic stability is critical to organismal survival. A failure to maintain this stability leads to deleterious events such as mutagenesis, chromosomal rearrangements, gene amplifications or deletions, and the gain or loss of entire chromosomes. These events reduce the fitness and may even endanger the life of unicellular organisms while leading to developmental abnormalities and tumorigenesis in metazoans. Selective pressure from these DNA insults has resulted in the evolution of a higher-order regulatory pathway referred to as the DNA-damage response (DDR), which emerged to coordinate repair processes both by directing the types of repair to be used for particular lesions and by coordinating this repair with other cellular events such as cell-cycle progression (1).The eukaryotic DDR consists of two central protein kinases of the PIKK family, ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) (3, 4). These proteins participate in sensing common intermediates in DNA repair, such as double-strand breaks or stalled replication forks, and thereupon phosphorylate downstream effectors to promote appropriate repair and cell-cycle coordination. The outlines of these pathways were originally established largely through studies in budding and fission yeast (1) and subsequently were expanded in mammals. In budding yeast, Tel1 (ATM homolog) and MEC1 (ATR homolog) carry out the bulk of the DNA-damage signaling. Unlike ATM in mammals, Tel1 has a relatively minor role in the budding yeast DDR (5). Downstream of these...

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“…Interestingly, Mec1 and Rad53 uncoupling also occurs in normal S phase, where Mec1, but not Rad53, is highly active. 85 It will be interesting to explore whether this uncoupling also requires the complex containing Slx4-Rtt107-Dpb11.…”

Section: The Rtt107 and Slx4 Association And Its Functions In Conjuncmentioning

confidence: 99%

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

2016

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Genome maintenance requires coordinated actions of diverse DNA metabolism processes. Scaffolding proteins, such as those containing multiple BRCT domains, can influence these processes by collaborating with numerous partners. The best-studied examples of multi-BRCT scaffolds are the budding yeast Dpb11 and its homologues in other organisms, which regulate DNA replication, repair, and damage checkpoints. Recent studies have shed light on another group of multi-BRCT scaffolds, including Rtt107 in budding yeast and related proteins in other organisms. These proteins also influence several DNA metabolism pathways, though they use strategies unlike those employed by the Dpb11 family of proteins. Yet, at the same time, these 2 classes of multi-BRCT proteins can collaborate under specific situations. This review summarizes recent advances in our understanding of how these multi-BRCT proteins function in distinct manners and how they collaborate, with a focus on Dpb11 and Rtt107.

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Phosphoproteomics Reveals Distinct Modes of Mec1/ATR Signaling during DNA Replication

Cited by 40 publications

References 0 publications

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

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

Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

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