Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase

Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase. damage and promotes apoptosis in CPT-treated cells. Taken together our results suggest that Stau2 is an anti-apoptotic protein that could be involved in DNA replication and/or maintenance of genome integrity and that its expression is regulated by E2F1 via the ATR signaling pathway. INTRODUCTION Chromosomal DNA is constantly exposed to endogenous and exogenous mutagens (1) that induce DNA damage with attendant genotoxic consequences including cell death, NH2-PEG3-C1-Boc mutagenesis and carcinogenesis (2). Therefore, to maintain genomic integrity, eukaryotic cells have evolved a finely-tuned global response, termed the DNA damage response (DDR), consisting of DNA damage detection leading to activation of signal transduction cascades that mediate reversible periods of cell cycle arrest and DNA repair (3,4). Alternatively, when repair pathways fail or become overwhelmed, or if cells are able to re-enter the growth cycle before repair is completed, mechanisms of irreversible growth arrest (senescence) or programmed cell death (apoptosis) are initiated (3). Senescence and apoptosis constitute powerful tumor-suppressive mechanisms that, respectively, completely forestall proliferation of, or destroy, NH2-PEG3-C1-Boc severely genetically-damaged cancer-prone cells. DDR pathways involve a preeminent contribution by the phosphoinositide 3-kinase related kinases, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-activated protein kinase (DNA-PK) (1,2). During genotoxic stress these enzymes phosphorylate hundreds of substrates either alone, or through the intermediacy of the downstream effector kinases checkpoint kinase 1 (CHEK1) and checkpoint kinase 2 (CHEK2) NH2-PEG3-C1-Boc activated primarily by ATR and ATM, respectively. Among other effects, this culminates in stimulation of transcription factors such as p53, E2F1 and NF-B which in turn positively and/or negatively regulate DDR gene expression. The DDR is differentially regulated depending on the type of DNA damage sustained by cells (1,2,5). Specifically, DNA double-strand breaks (DSBs) engender rapid activation NH2-PEG3-C1-Boc of the ATM and DNA-PK pathways (6) whereas DNA adducts that induce replicative stress by blocking the progression of DNA polymerases trigger rapid activation of the ATR pathway (7). Moreover, stalled replication forks may eventually collapse leading to DSB formation, and thus initial activation of ATR signaling can be followed by activation of ATM a number of hours later (8). Similarly, DSB formation initially sensed by ATM signaling is followed later during the repair process by DNA end resection, which generates RPA-coated single stranded overhangs leading to ATR activation (1,2,6). In any case, the mechanisms by which cells decide to induce programs leading to either cell cycle arrest/DNA repair or senescence/apoptosis are not entirely clear; however the balance between levels of pro- and anti-apoptotic proteins, mediated in large part by transcription factors such as p53, E2F1 and NF-B, lie at the heart of the decision (3,9C12). For example, E2F1-mediated activation of p53 results primarily in p53-dependent apoptosis rather than growth arrest (13C15). Indeed, certain critical proteins, many of which are transcription factors, can integrate diverse signals modulated by levels of DNA damage thereby finely tuning the equilibrium of pro- versus anti-apoptotic protein expression. High-throughput genomic/proteomic approaches have revealed RNA-binding proteins, as well as proteins implicated in RNA processing and post-transcriptional mRNA regulation, as putative novel regulators of the DDR (16C19). We thus became interested in the possibility of a potential role for Stau2 in the DDR. Stau2 is a double-stranded RNA-binding protein that associates with RNA secondary structures (20,21). The Stau2 gene, through differential splicing, generates at least four isoforms varying at Rabbit Polyclonal to ZNF174 their N- and/or C-termini. Stau2 is a component of ribonucleoprotein complexes (20,22,23) involved in mRNA transport (20,21,24), differential splicing (25), translation (26,27) and mRNA decay (28). In mammals, downregulation of this protein impairs mRNA transport to neuronal dendrites, causes dendritic spine defects and prevents long-term depression of hippocampal neurons (21,24,26). In zebrafish, Stau2 is required for survival and migration of.