In response to genotoxic stress, a transient arrest in cell cycle progression enforced by the DNA damage checkpoint (DDC) signaling pathway positively contributes to genome maintenance1. Because hyperactivated DDC can lead to a persistent and detrimental cell cycle arrest2,3, cells must tightly regulate the activity of DDC kinases. Despite their importance, the mechanisms for monitoring and modulating DDC signaling are not fully understood. Here we show that DNA repair scaffolding proteins Slx4 and Rtt107 prevent lesions generated during DNA replication from aberrantly hyperactivating DDC signaling in Saccharomyces cerevisiae. Upon replication stress, cells lacking Slx4 or Rtt107 exhibit hyperactivation of the downstream DDC kinase Rad53 while activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phospho-H2A, two positive regulators of Rad9-dependent Rad53 activation. Reduction of DDC signaling by hypomorphic mutations in RAD53 and H2A rescue the hyper-sensitivity of slx4Δ or rtt107Δ cells to replication stress. We propose that the Slx4-Rtt107 complex modulates Rad53 activation via a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings reveal that DDC signaling is monitored and modulated through the direct action of DNA repair factors.

Local cycling of LOK/SLK-dependent phosphorylation of ezrin is required for its apical localization and for microvillus formation.

The DNA damage checkpoint kinase Mec1(ATR) is critical for maintaining the integrity of replication forks. Though it has been proposed to promote fork repair, the mechanisms by which Mec1 regulates DNA repair factors remain unclear. Here, we found that Mec1 mediates a key interaction between the fork protein Dpb11 and the DNA repair scaffolds Slx4-Rtt107 to regulate replication stress response. Dissection of the molecular basis of the interaction reveals that Slx4 and Rtt107 jointly bind Dpb11 and that Slx4 phosphorylation is required. Mutation of Mec1 phosphorylation sites in Slx4 disrupts its interaction with Dpb11 and compromises the cellular response to replisomes blocked by DNA alkylation. Multiple fork repair factors associate with Rtt107 or Slx4, supporting that Mec1-dependent assembly of the Rtt107-Slx4-Dpb11 complex functions to coordinate fork repair. Our results unveil how Mec1 regulates the Slx4 and Rtt107 scaffolds and establish a mechanistic link between DNA damage signaling and fork repair.

The first steps of invasion and metastasis include the dissociation of adherens junctions and the induction of migratory phenotype, through a program that resembles epithelial-mesenchymal transition (EMT). The L1 cell adhesion molecule, which is normally found primarily in the brain, was recently shown to be expressed in different types of cancer and to have tumor-promoting activity. We now find that L1 mediates EMT-like events in MCF7 breast carcinoma cells. MCF7 predominantly expresses the nonneuronal isoform of L1, as do 16 of 17 other cell lines derived from different types of cancer. L1 protein expression in MCF7 cells, which form E-cadherin-containing adherens junctions, is inversely related to cell density. Analysis of MCF7 cells with overexpression or knockdown of nonneuronal L1 isoform revealed that L1 expression leads to the disruption of adherens junctions and increases B-catenin transcriptional activity. As a result, L1 expression promotes the scattering of epithelial cells from compact colonies. Expression of the fulllength L1 protein, but not of its soluble extracellular moiety, increases the motility of the MCF7 epithelial monolayer in a wound-healing assay, in which L1 expression is preferentially observed and required in cells leading the movement of the monolayer. Based on these results, we propose a model for the role of L1 as a trigger of EMT-like events in transformed epithelial cells.

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.