The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.Cells are highly responsive to their environment, especially DNA damaging agents. Damaged DNA in cells is rapidly sensed and turned into signals by the DNA damage checkpoint to control many processes, including cell cycle progression, DNA replication and repair, and gene transcription (1). The DNA damage checkpoint consists of several evolutionarily conserved protein kinases (2, 3). Understanding the function of the DNA damage checkpoint requires knowledge of their in vivo substrates. Although the regulation of DNA damage checkpoint kinases has been studied extensively, the knowledge of their in vivo substrates is limited. This can be attributed to the lack of suitable technology to detect low abundant phosphorylation in cells. With the use of stable isotope labeling, the advancement of high mass resolution mass spectrometry (MS), 3 and the recent development of analytical and computational tools by many laboratories (4 -9), changes in low abundant and regulatory phosphorylation in cells are increasingly detected. Combined with the use of genetics, in vivo kinase substrates have been identified using a quantitative mass spectrometry approach (10).In the yeast Saccharomyces cerevisiae, Mec1 and Tel1, homologs of the mammalian ATR and ATM kinase, respectively, function at the top of the signal transduction cascade in the DNA damage checkpoint (1-3). Mec1 is primarily responsible for the activation of downstream checkpoint kinases including Rad53 (11,12), whereas Tel1 has a more prominent role in regulating telomere length (13). Interestingly, deletion of both MEC1 and TEL1 leads to a synergistic increase in gross chromosomal rearrangements, indicating their redundant role in genome maintenance (14, 15). Mec1 is recruited to the site of DNA damage via replication protein A (RPA) that coats singlestranded DNA. Tel1 on the other hand is recruited by the Mre11-Rad50-Xrs2 complex, which recognizes DNA doublestranded breaks (DSBs) (16 -19). Importantly, DNA DSBs in cells undergo 5Ј to 3Ј resection to generate 3Ј single-...

Summary Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in non-neuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity.

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Post-translational modification by SUMO (small ubiquitin-like modifier) plays important but still poorly understood regulatory roles in eukaryotic cells, including as a signal for ubiquitination by SUMO targeted ubiquitin ligases (STUbLs). Here, we delineate the molecular mechanisms for SUMO-dependent control of ribosomal DNA (rDNA) silencing through the opposing actions of a STUbL (Slx5:Slx8) and a SUMO isopeptidase (Ulp2). We identify a conserved region in the Ulp2 C terminus that mediates its specificity for rDNA-associated proteins and show that this region binds directly to the rDNA-associated protein Csm1. Two crystal structures show that Csm1 interacts with Ulp2 and one of its substrates, the rDNA silencing protein Tof2, through adjacent conserved interfaces in its C-terminal domain. Disrupting Csm1's interaction with either Ulp2 or Tof2 dramatically reduces rDNA silencing and causes a marked drop in Tof2 abundance, suggesting that Ulp2 promotes rDNA silencing by opposing STUbL-mediated degradation of silencing proteins. Tof2 abundance is rescued by deletion of the STUbL or disruption of its SUMO-interacting motifs, confirming that Tof2 is targeted for degradation in a SUMO- and STUbL-dependent manner. Overall, our results demonstrate how the opposing actions of a localized SUMO isopeptidase and a STUbL regulate rDNA silencing by controlling the abundance of a key rDNA silencing protein, Tof2.