Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated-and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATRdependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.

DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.

The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to facilitate ATR activation in a substrateand dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/ breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.

The Mre11/Rad50/Nbs1 complex (MRN) plays an essential role in the S-phase checkpoint. Cells derived from patients with Nijmegen breakage syndrome and ataxia telangiectasia-like disorder undergo radioresistant DNA synthesis (RDS), failing to suppress DNA replication in response to ionizing radiation (IR). How MRN affects DNA replication to control the S-phase checkpoint, however, remains unclear. We demonstrate that MRN directly interacts with replication protein A (RPA) in unperturbed cells and that the interaction is regulated by cyclin-dependent kinases. We also show that this interaction is needed for MRN to correctly localize to replication centers. Abolishing the interaction of Mre11 with RPA leads to pronounced RDS without affecting phosphorylation of Nbs1 or SMC1 following IR. Moreover, MRN is recruited to sites at or adjacent to replication origins by RPA and acts there to inhibit new origin firing upon IR. These studies suggest a direct role of MRN at origin-proximal sites to control DNA replication initiation in response to DNA damage, thereby providing an important mechanism underlying the intra-S-phase checkpoint in mammalian cells.The Mre11/Rad50/Nbs1 complex (MRN) participates in multiple pathways to maintain genome stability (1, 10, 55). In humans, hypomorphic mutations in NBS1 and MRE11 lead to Nijmegen breakage syndrome (NBS) and ataxia-telangiectasialike disorder (ATLD), respectively (7,41,54,64). Both NBS and ATLD patients show developmental defects, immunodeficiency, and a high incidence of cancer, phenotypically similar to the ATM-deficient disorder ataxia-telangiectasia (AT) (53, 60). In addition, cells from NBS and ATLD patients are sensitive to radiation, exhibit genome instability, and have a defective S-phase checkpoint.The phenotypic resemblance of NBS, ATLD, and AT cells implies that MRN and ATM participate in similar biological pathways. This is supported by recent findings that MRN acts both upstream and downstream of ATM to mediate the damage response when double-strand breaks (DSBs) are generated (32). MRN migrates to DSB sites immediately after damage in an ATM-independent manner (44, 47) and is required for ATM activation, especially in response to low doses of ionizing radiation (IR) (6,8,25,35,63). MRN is also a direct substrate of ATM. Multiple ATM phosphorylation sites on Nbs1 have been identified, and these phosphorylation events play important roles in mediating the intra-S and G 2 /M checkpoints, although the mechanism by which Nbs1 phosphorylation regulates these pathways is not well defined (8,22,36,69,75).Mre11 is the core of the MRN complex and interacts with both Rad50 and Nbs1 (24, 34, 62). Mre11 carries 3Ј to 5Ј exonuclease activity and single-strand endonuclease activity and acts on various types of DSB ends and hairpins (49, 61). These nuclease activities are believed to be involved in processing of DNA ends for DSB repair (18,37,50). In multiple organisms, it has been shown that MRN is essential for DNA repair by homologous recombination (27,59,70).The radioresi...

The prevalence of intestinal inflammatory diseases is increasing, and pharmacologic agents for intervention are currently limited. Preserving epithelial tight junction (TJ) integrity and preventing underlying immune cell activation by intestinal bacteria are key targets for abrogating the perpetual inflammatory cycle that plagues these diseases. Phytonutrients have shown promise for their ability to reduce cellular inflammation, but the extent of their efficacy in an intestinal model of inflammation is not well understood. Here we hypothesized that Spectra7 (S7), a novel phytonutrient derived from extracts rich in curcuminoids and catechins, would reduce immune cell inflammation and preserve TJ integrity in an in vitro co‐culture model of intestinal inflammation. We further hypothesized that a curcumin containing formulation (S7‐C) would be more effective at preserving TJ integrity than that of its metabolite, tetrahydrocurcumin (S7‐THC). An in vitro intestinal co‐culture model was established by seeding Caco‐2 epithelial cells on semi‐permeable transwell inserts 21 days prior to the addition of RAW264.7 macrophages in the basolateral chamber. Macrophages were next stimulated with 10 ng/ml lipopolysaccharide (LPS) to induce inflammation, and subsequent TJ disruption in the co‐cultured Caco‐2 cells was assessed by transepithelial electrical resistance (TEER) using epithelial ohmmeter chopstick electrodes. We found that administration of S7‐THC containing 1–5 μM THC produced dose dependent mitigation of LPS‐induced decreases in TEER and approached the efficacy of the pharmacologic agent budesonide. However, to our surprise, S7‐C at 5 μM curcumin was unable to preserve TEER, suggesting that the specific combination of phytonutrients is important for preventing inflammation‐induced TJ disruption. We also found that, though apical application (Caco‐2 only) of budesonide was sufficient for preserving TEER in our model, S7‐THC required both apical (Caco‐2) and basolateral (RAW264.7) treatment, suggesting that reducing macrophage inflammation is important for limiting epithelial TJ disruption in this context. Interestingly, S7‐C was more effective than S7‐THC or budesonide at reducing inflammatory basolateral nitric oxide (NO) production as determined by the Griess assay. This suggests that, though S7‐C more effectively reduces this aspect of inflammation, another inflammatory mediator is responsible for conveying TJ disruption and is governed differentially by S7‐THC. Further support of this comes from our finding that S7‐THC, but not S7‐C, ameliorated the LPS‐induced increase in myosin light chain kinase (MLCK) expression in Caco‐2 cells as determined by Western blot. Together, these findings suggest that phytonutrients such as S7‐THC have prophylactic potential in the preservation of TJ integrity, and the specific composition of these phytonutrients matters. Support or Funding Information Olivet Nazarene University Honors Program, Pence‐Boyce Undergraduate Summer Research Experience, and the Hippenhammer Faculty Grant

Uemura's proposed biosynthetic pathway of symbioimine was demonstrated by microwave-assisted intramolecular Diels-Alder reaction of a trans-enone precursor.

Unbalanced or prolonged inflammation contributes to the pathogenesis of many conditions such as diabetes, arthritis, atherosclerosis and cancer to name a few. At the cellular level, macrophages of the innate immune system are central players that become inflamed in an M1‐state upon encountering pathogen, and, following clearance, shift in activation to an anti‐inflammatory (M2) state to aid in resolution of inflammation. Unfortunately, chronic diseases are promoted and exacerbated by M1‐macrophages that have become “stuck” in the on position. Spectra7 (S7) is a novel phytonutrient supplement containing extracts rich in curcuminoids, epigallocatechin gallate (EGCG), catechins, chlorogenic acid and anthocyanins. While these phytonutrients have individually demonstrated anti‐inflammatory properties in previous research, it is not known if the combination of these in S7 is effective at reducing M1‐macrophage activation. Here we tested the hypothesis that S7 would reduce LPS‐induced M1‐macrophage activation, and that the combination of these phytonutrients would provide greater inhibition than the sum of the individual components. Hypothesizing that the curcuminoids would exert the greatest effect on M1 activation, we further tested two formulations of S7 with either curcumin (S7‐C) or its metabolite tetrahydrocurcumin (S7‐THC). Phytonutrients individually or in combination were tested for their ability to alter LPS‐induced M1 activation in RAW264.7 macrophages. We found that both S7‐THC and S7‐C exhibited strong dose‐dependent inhibition of nitric oxide (NO) production as assessed by the Griess assay. Moreover, this effect was largely attributed to EGCG in the green tea extract (GTE) and curcuminoids in the turmeric extract (TE). Combinations of phytonutrients containing 5–10 μM curcumin exerted the greatest effect. Interestingly, while the other five components of S7 (green coffee bean extract, blueberries, whole tart cherries, broccoli and kale) did not inhibit NO production, they did enhance the ability of GTE and TE in inhibiting M1‐activation. This suggests that the combination of individual ingredients together in S7‐THC/C exhibit synergistic interactions. Determination of inflammatory gene expression revealed differential regulation between S7‐C, S7‐THC and the corticosteroid budesonide. While all three inhibited LPS‐induced NO production, only S7‐C and budesonide decreased expression of inducible nitric oxide synthase (iNOS), suggesting posttranscriptional regulation was also a factor. IFN‐β and IL‐6 expression were also inhibited by S7‐C and budesonide, but S7‐THC paradoxically prolonged LPS‐induced expression at 12 and 24 hours, suggesting delayed negative feedback inhibition. Lastly, we also found that S7‐C and budesonide, but not S7‐THC, were able to prevent cellular and mitochondrial reactive oxygen species (ROS) as assessed by DCFDA and MitoSOX fluorescence respectively. Together, these data support continued investigation of natural alternatives, like S7‐THC/C, to pharmacologic agents for their ability...