ABSTRACT
DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.
Subject(s)
Chromosomes, Human/genetics , DNA Adducts/genetics , DNA Primase/physiology , DNA Replication , DNA-Directed DNA Polymerase/physiology , Multifunctional Enzymes/physiology , Amino Acid Sequence , Animals , Cell Proliferation , Cell Survival , Chickens , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA Damage , DNA Primase/chemistry , DNA, Single-Stranded/chemistry , DNA-Directed DNA Polymerase/chemistry , G2 Phase Cell Cycle Checkpoints , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Mice, Knockout , Molecular Sequence Data , Multifunctional Enzymes/chemistry , Ultraviolet Rays , XenopusABSTRACT
Nuclear factor of activated T cells (NFAT) proteins are a group of Ca(2+)-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17- and IL-10-producing cells after differentiation under Th17 conditions-this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca(2+)/NFAT signaling to lymphoproliferation and autoimmune disease.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , NFATC Transcription Factors/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Base Sequence , Binding Sites/genetics , Calcineurin/metabolism , Calcium Signaling , Casein Kinase I/metabolism , Cell Differentiation , Central Nervous System/immunology , Central Nervous System/metabolism , Central Nervous System/pathology , DNA Primers/genetics , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Forkhead Transcription Factors/metabolism , Interleukin-10/biosynthesis , Interleukin-10/genetics , Interleukin-17/biosynthesis , Interleukin-17/genetics , Mice , Mice, Transgenic , Molecular Sequence Data , NFATC Transcription Factors/genetics , Protein Engineering , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , T-Lymphocytes/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.
