The checkpoint response to replication stress.
ABSTRACT Genome instability is a hallmark of cancer cells, and defective DNA replication, repair and recombination have been linked to its etiology. Increasing evidence suggests that proteins influencing S-phase processes such as replication fork movement and stability, repair events and replication completion, have significant roles in maintaining genome stability. DNA damage and replication stress activate a signal transduction cascade, often referred to as the checkpoint response. A central goal of the replication checkpoint is to maintain the integrity of the replication forks while facilitating replication completion and DNA repair and coordinating these events with cell cycle transitions. Progression through the cell cycle in spite of defective or incomplete DNA synthesis or unrepaired DNA lesions may result in broken chromosomes, genome aberrations, and an accumulation of mutations. In this review we discuss the multiple roles of the replication checkpoint during replication and in response to replication stress, as well as the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.
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ABSTRACT: Ribosomal biogenesis involves processing of pre-rRNA in assembly with ribosomal proteins (RPs). Deficiency of some RPs impairs processing and causes Diamond Blackfan Anemia (DBA) associated with anemia, congenital malformations, and cancer. p53 mediates many features of DBA but the mechanism of p53 activation remains unclear. Another hallmark of DBA is upregulation of adenosine deaminase (ADA) suggesting changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis consistent with the need to break and replace the faulty rRNA. We also found upregulation of dNTP synthesis, a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM/CHK1/2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion, and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements may be beneficial in DBA.Disease Models and Mechanisms 05/2014; · 4.96 Impact Factor
Article: Genetic instability is prevented by Mrc1-dependent spatio-temporal separation of replicative and repair activities of homologous recombination: Homologous recombination tolerates replicative stress by Mrc1-regulated replication and repair activities operating at S and G2 in distinct subnuclear compartments.[Show abstract] [Hide abstract]
ABSTRACT: Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability.BioEssays 02/2014; · 5.42 Impact Factor
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ABSTRACT: Guanine nucleotide exchange factors (GEFs) control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is re-localized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence (NLS) at the N-terminus of Rgf1p, while release into the cytoplasm requires two leucine-rich nuclear export sequences (NES) at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and on the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes 9 Ser potential phosphorylation Cds1p sites for Ala fails to accumulate in the nucleus in response to replication stress and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologs of Rgf1p in budding yeast and higher eukaryotes.Molecular biology of the cell 01/2014; · 5.98 Impact Factor