Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair. T he cellular response to DNA damage is initiated by the sensing of structural alterations in DNA that culminates in the activation of phosphoinositide-3-kinase-related protein kinases (PIKKs) that include the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases (1). With the help of mediators, ATM and ATR subsequently signal downstream to activate effector kinases checkpoint 1 (CHK1) and checkpoint 2 (CHK2), leading to transcriptional induction, cell-cycle arrest, DNA repair, senescence, or apoptosis. This DNA damage response induces the sequential recruitment of an extensive network of proteins to the sites of damage. For example, in response to double-strand breaks (DSBs), ATM phosphorylates histone H2AX adjacent to the break to initiate a H2AX-dependent concentration of proteins involved in the DNA damage response, such as mediator of DNA damage checkpoint protein 1 (MDC1), which recruits additional molecules of the ATM kinase. This recruitment effectively initiates a positive feedback loop that promotes the spread of γH2AX-flanking DSBs (2). Phosphorylation of MDC1 by ATM creates a motif that is recognized by the ubiquitin ligase ring finger 8 (RNF8) (3-6) that, with the help of ring finger 168 (RNF168), catalyzes the formation of lysine 63 (K63)-linked polyubiquitin chains that ultimately recruit the breast cancer 1 (BRCA1) A complex containing receptor-associated protein 80 (RAP80), Abraxas, BRCA1, new component of the BRCA1 A complex (NBA1), and BRCA1/BRCA2-containing complex, subunit 3 (BRCC36) (3-10) as well as p53 binding protein 1 (53BP1) and RAD18 homolog (RAD18) (3-8, 11).Several factors, such as Nijmegen breakage syndrome 1 (NBS1), 53BP1, and BRCA1, are recruited to the sites of damage in an H2AX-independent manner (12). However, these interactions appear to be more transient and may play a role as an initial response to DNA damage that is distinct from the extended association of factors via γH2AX. Several additional pathways also have been shown to direct the recruitment of vario...

The abundance of cellular proteins is determined largely by the rate of transcription and translation coupled with the stability of individual proteins. Although we know a great deal about global transcript abundance, little is known about global protein stability. We present a highly parallel multiplexing strategy to monitor protein turnover on a global scale by coupling flow cytometry with microarray technology to track the stability of individual proteins within a complex mixture. We demonstrated the feasibility of this approach by measuring the stability of approximately 8000 human proteins and identifying proteasome substrates. The technology provides a general platform for proteome-scale analysis of protein turnover under various physiological and disease conditions.

Tipin is a mammalian protein that interacts with Timeless, which plays a role in DNA damage checkpoint responses. Here, we show that Tipin is a nuclear protein that associates with the replicative helicase and protects cells against genotoxic agents. Tipin is required for efficient cell cycle arrest in response to DNA damage, and depletion of Tipin renders cells sensitive to ionizing radiation as well as replication stress. Loss of Tipin results in spontaneous ␥-H2AX foci, a marker for DNA double-strand breaks. We find that Tipin and Timeless form a complex that maintains the level of both proteins in cells and that the loss of either one will lead to the loss of the interacting partner. This observation explains the similar checkpoint phenotypes observed in both Tipin-and Timeless-depleted cells.DNA damage ͉ S phase ͉ cell cycle control ͉ circadian rhythms

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here we report the characterization of Torin2, a second generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pM with approximately 800-fold selectivity for cellular mTOR versus PI3K. Torin2 also exhibited potent biochemical and cellular activity against PIKK family kinases including ATM (EC50 28 nM), ATR (EC50 35 nM) and DNA-PK (EC50 118 nM) (PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with MEK inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncological settings where mTOR signaling has a pathogenic role.

The Mediator complex associates with eukaryotic RNA polymerase (Pol) II and is recruited to transcriptional enhancers by activator proteins. It is believed that Mediator is a general component of the Pol II machinery that is crucial to connect enhancer-bound activators to basic transcription factors. However, we show that Mediator does not detectably associate with many highly active Pol II promoters in yeast cells. Furthermore, in response to stress conditions, Mediator association is not directly related to Pol II association and in some cases is not detectable at highly activated promoters. Thus, Mediator is recruited to enhancers in an activator-specific manner, and it does not seem to be a stoichiometric component of the basic Pol II machinery in vivo. Mediator is recruited by many activators involved in stress responses, but not by the major activators that function under optimal conditions.

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...

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxiatelangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.Initiation of DNA replication in eukaryotes occurs in two major steps, which are strictly coordinated with cell cycle progression. The first step takes place during the early G 1 phase and involves formation of a prereplication complex (pre-RC) at the origin of DNA replication. The pre-RC consists of the origin recognition complex, Cdt1, Cdc6, and MCM2-7, the hexameric minichromosome maintenance protein, and presumed DNA helicase (for reviews, see references 4 and 42). The second step occurs at the G 1 /S transition and involves conversion of the pre-RC into an initiation complex (IC) through the sequential binding of Cdc45, single-strand DNA binding protein RPA, and DNA polymerase ␣/primase. Cdc45 loading onto the pre-RC is a key step in IC formation, which depends on MCM2-7 (4), MCM10 (48), and two protein kinases, the cyclin-dependent kinase 2 (Cdk2)-cyclin E and Cdc7-Dbf4 (4), as well as on Xmus101/Cut5 (19, 45). Furthermore, Cdc45 loading in yeast depends on Sld3, which forms a complex with Cdc45 (23, 34). Following initiation, MCM2-7 and Cdc45 form a tight complex on chromatin, which is thought to carry out DNA unwinding (31, 35). Recently, GINS, an essential replication factor in Xenopus and Saccharomyces cerevisiae, has been identified, which consists of four subunits (24,27,43). In Xenopus, chromatin loading of GINS depends on pre-RC assembly, Mus101, and Cdk2-cyclin E. In add...

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘Candidatus Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.