Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase e Fabio Puddu, Gabriele Piergiovanni¤, Paolo Plevani*, Marco Muzi-Falconi* Department of Biomolecular Sciences and Biotechnology, Universita` degli Studi di Milano, Milano, Italy Abstract Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Ddpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non- essential subunit of DNA polymerase e (Pole) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Pole. Citation: Puddu F, Piergiovanni G, Plevani P, Muzi-Falconi M (2011) Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase e. PLoS Genet 7(3): e1002022. doi:10.1371/journal.pgen.1002022 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received November 10, 2010; Accepted January 20, 2011; Published March 17, 2011 Copyright: 2011 Puddu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from MIUR, AIRC, Fondazione Cariplo, and the European Union FP6 Integrated Project DNA repair contract number 512113 to to PP and MM-F. FP was supported through a fellowship from Fodazione A. Buzzati Traverso. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marco.muzifalconi@unimi.it (MM-F); paolo.plevani@unimi.it (PP) ¤ Current address: Genome Stability Unit, Clare Hall Laboratories, London Research Institute, London, United Kingdom Introduction The DNA replication machinery can experience various types of stress during S phase. This can happen when the replisome encounters DNA lesions that hinder its progression, while traversing slow replication zones corresponding to genomic regions difficult to replicate [1] or when encountering replication fork barriers [2]. Replication stress can also be induced by inhibiting ribonucleotide reductase (RNR) with hydroxyurea, which causes a global replication arrest by reducing the dNTPs pools [3]. Under replication stress conditions, eukaryotic cells trigger a signaling cascade, known as the replication checkpoint, which, in budding yeast, culminates with the phosphorylation of Rad53 [4]. This protein kinase is essential for the activation of the molecular mechanisms required to cope with replication arrest: it promotes stabilization of stalled replication forks and allows DNA replication re-start after removal of the blocking agent [5,6,7,8]. Rad53 is also responsible for inducing the transcription of RNR genes by inhibiting the transcriptional repressor Crt1 and promoting the degradation of the RNR inhibitor Sml1 [9,10]. Finally, Rad53 prevents the firing of late replication origins [11] and restrains spindle elongation thus preventing mitosis [12,13,14]. The DNA damage and replication checkpoints are genetically distinct pathways; however, they are partially overlapping since they share several of the factors involved. In fact, replication stress activates Mec1, the same apical kinase triggered by DNA damage, which is recruited to RPA-covered ssDNA by its binding partner Ddc2 [15]. After damage, Mec1 phosphorylates the Rad9 adaptor protein, which has been loaded onto DNA via chromatin- dependent and -independent pathways: the former requiring methylation of H3-K79 and the latter depending on the 9-1-1 complex and Dpb11 [16,17,18,19,20]. Phosphorylated Rad9, in turn, recruits Rad53, which becomes hyperphosphorylated in a Mec1-dependent manner. Differently, in the case of HU-induced checkpoint activation, the Rad9 adaptor protein is dispensable and its function is performed by Mrc1, a constitutive member of the replisome complex [21,22]. It is now clear that following genotoxin treatments, primary lesions are generally recognized by specific repair factors that process them to generate ssDNA regions, which elicit the DNA damage response. On the other hand, the actual mechanism acting in the activation of the replication stress response is poorly understood. In budding yeast, it has been suggested that replication proteins may be involved in sensing blocks of the replication fork. Indeed, in addition to Dpb11, the initiation factor Sld2/Drc1 and Pole itself are required for efficient checkpoint activation in response to HU treatment, although the correspond- ing mutants are only mildly sensitive to the drug [23,24,25]. Sld2 is an essential CDK1 target required for initiation of DNA replication. Its phosphorylation and subsequent interaction with Dpb11 is essential for the loading of Pole and the firing of PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1002022