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1.
Science ; 382(6675): eadf3208, 2023 12 08.
Article in English | MEDLINE | ID: mdl-38060659

ABSTRACT

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.


Subject(s)
Aging , MAP Kinase Kinase Kinase 3 , Obesity , Reactive Oxygen Species , Ribosomes , Stress, Physiological , Animals , Mice , Aging/metabolism , MAP Kinase Kinase Kinase 3/genetics , MAP Kinase Kinase Kinase 3/metabolism , Obesity/metabolism , Protein Biosynthesis , Reactive Oxygen Species/metabolism , Ribosomes/metabolism , Zebrafish , Mice, Knockout
2.
Cell Death Dis ; 14(7): 467, 2023 07 26.
Article in English | MEDLINE | ID: mdl-37495584

ABSTRACT

Impairment of protein translation can cause stalling and collision of ribosomes and is a signal for the activation of ribosomal surveillance and rescue pathways. Despite clear evidence that ribosome collision occurs stochastically at a cellular and organismal level, physiologically relevant sources of such aberrations are poorly understood. Here we show that a burst of the cellular signaling molecule nitric oxide (NO) reduces translational activity and causes ribosome collision in human cell lines. This is accompanied by activation of the ribotoxic stress response, resulting in ZAKα-mediated activation of p38 and JNK kinases. In addition, NO production is associated with ZNF598-mediated ubiquitination of the ribosomal protein RPS10 and GCN2-mediated activation of the integrated stress response, which are well-described responses to the collision of ribosomes. In sum, our work implicates a novel role of NO as an inducer of ribosome collision and activation of ribosomal surveillance mechanisms in human cells.


Subject(s)
Nitric Oxide , Ribosomes , Humans , Nitric Oxide/metabolism , Ribosomes/metabolism , Protein Biosynthesis , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ubiquitination , Carrier Proteins/metabolism
3.
Cells ; 12(6)2023 03 22.
Article in English | MEDLINE | ID: mdl-36980309

ABSTRACT

The kinase ZAKα acts as the proximal sensor of translational impairment and ribotoxic stress, which results in the activation of the MAP kinases p38 and JNK. Despite recent insights into the functions and binding partners of individual protein domains in ZAKα, the mechanisms by which ZAKα binds ribosomes and becomes activated have remained elusive. Here, we highlight a short, thrice-repeated, and positively charged peptide motif as critical for the ribotoxic stress-sensing function of the Sensor (S) domain of ZAKα. We use this insight to demonstrate that the mutation of the SAM domain uncouples ZAKα activity from ribosome binding. Finally, we use 3D structural comparison to identify and functionally characterize an additional folded domain in ZAKα with structural homology to YEATS domains. These insights allow us to formulate a model for ribosome-templated ZAKα activation based on the re-organization of interactions between modular protein domains. In sum, our work both advances our understanding of the protein domains and 3D architecture of the ZAKα kinase and furthers our understanding of how the ribotoxic stress response is activated.


Subject(s)
Ribosomes , p38 Mitogen-Activated Protein Kinases , Ribosomes/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
4.
Antioxid Redox Signal ; 39(4-6): 336-350, 2023 08.
Article in English | MEDLINE | ID: mdl-36825529

ABSTRACT

Significance: Translation is an essential cellular process, and diverse signaling pathways have evolved to deal with problems arising during translation. Erroneous stalls and unresolved ribosome collisions are implicated in many pathologies, including neurodegeneration and metabolic dysregulation. Recent Advances: Many proteins involved in detection and clearance of stalled and collided ribosomes have been identified and studied in detail. Ribosome profiling techniques have revealed extensive and nonprogrammed ribosome stalling and leaky translation into the 3' untranslated regions of mRNAs. Impairment of protein synthesis has been linked to aging in yeast and mice. Critical Issues: Ribosomes act as sensors of cellular states, but the molecular mechanisms, as well as physiological relevance, remain understudied. Most of our current knowledge stems from work in yeast and simple multicellular organisms such as Caenorhabditis elegans, while we are only beginning to comprehend the role of ribosome surveillance in higher organisms. As an example, the ribotoxic stress response, a pathway responding to global translational stress, has been studied mostly in response to small translation inhibitors and ribotoxins, and has only recently been explored in physiological settings. This review focuses on ribosome-surveillance pathways and their importance for cell and tissue homeostasis upon naturally occurring insults such as oxidative stress, nutrient deprivation, and viral infections. Future Directions: A better insight into the physiological roles of ribosome-surveillance pathways and their crosstalk could lead to an improved understanding of human pathologies and aging. Antioxid. Redox Signal. 39, 336-350.


Subject(s)
Protein Biosynthesis , Saccharomyces cerevisiae , Humans , Animals , Mice , Saccharomyces cerevisiae/metabolism , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/metabolism , Oxidative Stress
5.
Cell Metab ; 34(12): 2036-2046.e8, 2022 12 06.
Article in English | MEDLINE | ID: mdl-36384144

ABSTRACT

Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.


Subject(s)
MAP Kinase Kinase Kinases , Protein Biosynthesis , Ribosomes , Stress, Physiological , Animals , Male , Mice , MAP Kinase Kinase Kinases/metabolism
6.
EMBO J ; 41(17): e111650, 2022 09 01.
Article in English | MEDLINE | ID: mdl-35899396

ABSTRACT

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.


Subject(s)
Mitogen-Activated Protein Kinases , Muscle, Skeletal , Animals , MAP Kinase Kinase Kinases , Mice , Mitogen-Activated Protein Kinases/metabolism , Muscle Contraction/physiology , Muscle, Skeletal/metabolism , Phosphorylation , Signal Transduction/physiology , p38 Mitogen-Activated Protein Kinases/genetics
7.
Int J Mol Sci ; 22(17)2021 Sep 04.
Article in English | MEDLINE | ID: mdl-34502507

ABSTRACT

p38 and c-Jun N-terninal kinase (JNK) are activated in response to acute stress and inflammatory signals. Through modification of a plethora of substrates, these kinases profoundly re-shape cellular physiology for the optimal response to a harmful environment and/or an inflammatory state. Here, we utilized phospho-proteomics to identify several hundred substrates for both kinases. Our results indicate that the scale of signaling from p38 and JNK are of a similar magnitude. Among the many new targets, we highlight the regulation of the transcriptional regulators grb10-interacting GYF protein 1 and 2 (GIGYF1/2) by p38-dependent MAP kinase-activated protein kinase 2 (MK2) phosphorylation and 14-3-3 binding. We also show that the Golgi apparatus contains numerous substrates, and is a major target for regulation by p38 and JNK. When activated, these kinases mediate structural rearrangement of the Golgi apparatus, which positively affects protein flux through the secretory system. Our work expands on our knowledge about p38 and JNK signaling with important biological ramifications.


Subject(s)
MAP Kinase Kinase 4/metabolism , Stress, Physiological/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , Carrier Proteins/metabolism , Cell Line, Tumor , Golgi Apparatus/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase 4/physiology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/physiology
8.
Mol Cell ; 78(4): 700-713.e7, 2020 05 21.
Article in English | MEDLINE | ID: mdl-32289254

ABSTRACT

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.


Subject(s)
Caenorhabditis elegans/growth & development , MAP Kinase Kinase Kinases/metabolism , Peptidyl Transferases/metabolism , RNA, Ribosomal, 18S/metabolism , Ribosomes/metabolism , Stress, Physiological , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Enzyme Activation , HeLa Cells , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/genetics , Protein Conformation , Protein Domains , RNA, Ribosomal, 18S/genetics , Sequence Homology , Signal Transduction
9.
Cell Rep ; 26(13): 3511-3521.e4, 2019 03 26.
Article in English | MEDLINE | ID: mdl-30917308

ABSTRACT

Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner. Similar to TTP, ZNF598 harbors three proline-rich motifs that bind the GYF domain of GIGYF1. RNA sequencing experiments showed that ZNF598 is required for the regulation of known TTP targets, including IL-8 and CSF2 mRNA. Furthermore, we demonstrate that ZNF598 binds to IL-8 mRNA, but not TNF mRNA. Collectively, our findings highlight that ZNF598 functions as an RBP that buffers the level of a range of mRNAs. We propose that ZNF598 is a TTP-like factor that can contribute to the regulation of the inflammatory potential of cytokine-producing cells.


Subject(s)
Carrier Proteins/metabolism , Inflammation/metabolism , RNA Processing, Post-Transcriptional , Signal Transduction , Tristetraprolin/metabolism , Amino Acid Motifs , Animals , Cell Line, Tumor , Cytokines/metabolism , Humans , Inflammation/genetics , Protein Binding , RNA, Messenger/metabolism
10.
Mol Cell ; 74(2): 254-267.e10, 2019 04 18.
Article in English | MEDLINE | ID: mdl-30824372

ABSTRACT

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.


Subject(s)
Positive Transcriptional Elongation Factor B/genetics , RNA Polymerase II/genetics , RNA-Binding Proteins/genetics , Transcription, Genetic , Apoptosis/genetics , Cell Survival/genetics , DNA Damage/genetics , HEK293 Cells , Humans , RNA, Long Noncoding/genetics , Ribonucleoproteins, Small Nuclear/genetics , p38 Mitogen-Activated Protein Kinases/genetics
11.
Nat Commun ; 6: 10075, 2015 Nov 30.
Article in English | MEDLINE | ID: mdl-26616734

ABSTRACT

Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress.


Subject(s)
14-3-3 Proteins/metabolism , Cell Cycle Proteins/metabolism , Centrioles/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microtubule Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , 14-3-3 Proteins/genetics , Cell Cycle Proteins/genetics , Centrioles/enzymology , Centrioles/genetics , Cytoskeletal Proteins , Humans , Intracellular Signaling Peptides and Proteins/genetics , Microtubule Proteins/genetics , Protein Binding , Protein Serine-Threonine Kinases/genetics , Signal Transduction , p38 Mitogen-Activated Protein Kinases/genetics
12.
Genes Dev ; 28(18): 1977-82, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25189701

ABSTRACT

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.


Subject(s)
14-3-3 Proteins/metabolism , DNA Damage/physiology , Exosome Multienzyme Ribonuclease Complex/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , RNA, Untranslated/metabolism , Ultraviolet Rays
15.
Genome Biol ; 12(8): R78, 2011 Aug 18.
Article in English | MEDLINE | ID: mdl-21851590

ABSTRACT

BACKGROUND: The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1 substrates have hitherto been identified. RESULTS: Here, we combine a chemical genetics approach with high-resolution mass spectrometry to identify novel Chk1 substrates and their phosphorylation sites. The list of targets produced reveals the potential impact of Chk1 function not only on processes where Chk1 was already known to be involved, but also on other key cellular events such as transcription, RNA splicing and cell fate determination. In addition, we validate and explore the phosphorylation of transcriptional co-repressor KAP1 Ser473 as a novel DNA-damage-induced Chk1 site. CONCLUSIONS: By providing a substantial set of potential Chk1 substrates, we present opportunities for studying unanticipated functions for Chk1 in controlling a wide range of cellular processes. We also refine the Chk1 consensus sequence, facilitating the future prediction of Chk1 target sites. In addition, our identification of KAP1 Ser473 phosphorylation as a robust readout for Chk1 activity could be used to explore the in vivo effects of Chk1 inhibitors that are being developed for clinical evaluation.


Subject(s)
Protein Kinases/genetics , Protein Kinases/metabolism , Proteomics/methods , Amino Acid Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Checkpoint Kinase 1 , Co-Repressor Proteins/genetics , Co-Repressor Proteins/metabolism , DNA Damage , DNA Replication , Humans , Mitosis , Molecular Sequence Data , Phosphorylation , Repressor Proteins/metabolism , Tripartite Motif-Containing Protein 28
16.
PLoS One ; 6(8): e23517, 2011.
Article in English | MEDLINE | ID: mdl-21858151

ABSTRACT

The DNA-damage checkpoint kinase Chk1 is essential in higher eukaryotes due to its role in maintaining genome stability in proliferating cells. CHK1 gene deletion is embryonically lethal, and Chk1 inhibition in replicating cells causes cell-cycle defects that eventually lead to perturbed replication and replication-fork collapse, thus generating endogenous DNA damage. What is the cause of replication-fork collapse when Chk1 is inactivated, however, remains poorly understood. Here, we show that generation of DNA double-strand breaks at replication forks when Chk1 activity is compromised relies on the DNA endonuclease complex Mus81/Eme1. Importantly, we show that Mus81/Eme1-dependent DNA damage--rather than a global increase in replication-fork stalling--is the cause of incomplete replication in Chk1-deficient cells. Consequently, Mus81/Eme1 depletion alleviates the S-phase progression defects associated with Chk1 deficiency, thereby increasing cell survival. Chk1-mediated protection of replication forks from Mus81/Eme1 even under otherwise unchallenged conditions is therefore vital to prevent uncontrolled fork collapse and ensure proper S-phase progression in human cells.


Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Protein Kinases/metabolism , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , Checkpoint Kinase 1 , Comet Assay , DNA/chemistry , DNA/genetics , DNA Breaks, Double-Stranded/drug effects , DNA Repair , DNA Replication , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Nucleic Acid Conformation , Protein Kinases/genetics , RNA Interference , S Phase , Thiophenes/pharmacology , Urea/analogs & derivatives , Urea/pharmacology
17.
Crit Rev Biochem Mol Biol ; 43(3): 221-38, 2008.
Article in English | MEDLINE | ID: mdl-18568848

ABSTRACT

Deinococcus radiodurans, one of the most radioresistant organisms known to date, is able to repair efficiently hundreds of DNA double- and single-strand breaks as well as other types of DNA damages promoted by ionizing or ultraviolet radiation. We review recent discoveries concerning several aspects of radioresistance and survival under high genotoxic stress. We discuss different hypotheses and possibilities that have been suggested to contribute to radioresistance and propose that D. radiodurans combines a variety of physiological tools that are tightly coordinated. A complex network of regulatory proteins may be discovered in the near future that might allow further understanding of radioresistance.


Subject(s)
Deinococcus/cytology , Deinococcus/radiation effects , DNA Repair/radiation effects , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Bacterial/radiation effects , Deinococcus/genetics , Deinococcus/metabolism , Desiccation , Genome, Bacterial/genetics , Genome, Bacterial/radiation effects , Microbial Viability/radiation effects
18.
BMC Mol Biol ; 8: 69, 2007 Aug 16.
Article in English | MEDLINE | ID: mdl-17705817

ABSTRACT

BACKGROUND: Enzymes involved in DNA metabolic events of the highly radioresistant bacterium Deinococcus radiodurans are currently examined to understand the mechanisms that protect and repair the Deinococcus radiodurans genome after extremely high doses of gamma-irradiation. Although several Deinococcus radiodurans DNA repair enzymes have been characterised, no biochemical data is available for DNA ligation and DNA endhealing enzymes of Deinococcus radiodurans so far. DNA ligases are necessary to seal broken DNA backbones during replication, repair and recombination. In addition, ionizing radiation frequently leaves DNA strand-breaks that are not feasible for ligation and thus require end-healing by a 5'-polynucleotide kinase or a 3'-phosphatase. We expect that DNA ligases and end-processing enzymes play an important role in Deinococcus radiodurans DNA strand-break repair. RESULTS: In this report, we describe the cloning and expression of a Deinococcus radiodurans DNA ligase in Escherichia coli. This enzyme efficiently catalyses DNA ligation in the presence of Mn(II) and NAD+ as cofactors and lysine 128 was found to be essential for its activity. We have also analysed a predicted second DNA ligase from Deinococcus radiodurans that is part of a putative DNA repair operon and shows sequence similarity to known ATP-dependent DNA ligases. We show that this enzyme possesses an adenylyltransferase activity using ATP, but is not functional as a DNA ligase by itself. Furthermore, we identified a 5'-polynucleotide kinase similar to human polynucleotide kinase that probably prepares DNA termini for subsequent ligation. CONCLUSION: Deinococcus radiodurans contains a standard bacterial DNA ligase that uses NAD+ as a cofactor. Its enzymatic properties are similar to E. coli DNA ligase except for its preference for Mn(II) as a metal cofactor. The function of a putative second DNA ligase remains unclear, but its adenylyltransferase activity classifies it as a member of the nucleotidyltransferase family. Characterization of another protein from the same operon revealed a 5'-polynucleotide kinase with a possible role in DNA strand-break repair.


Subject(s)
Bacterial Proteins/metabolism , DNA Ligases/metabolism , DNA Repair , DNA, Bacterial , Deinococcus/genetics , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Radiation, Ionizing , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , DNA Damage , DNA Ligases/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Bacterial/radiation effects , Deinococcus/metabolism , Humans , Manganese/metabolism , Molecular Sequence Data , NAD/metabolism , Polynucleotide 5'-Hydroxyl-Kinase/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment
19.
Mol Microbiol ; 60(1): 165-76, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16556228

ABSTRACT

Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure-modulated but not the canonical 3'-->5' exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure-specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure-modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolXDr is the first family X DNA polymerase that harbours an exonuclease activity. The wild-type protein, the glycine mutant and the two domains were expressed separately in DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.


Subject(s)
DNA-Directed DNA Polymerase , Deinococcus/radiation effects , Exonucleases/metabolism , Radiation Tolerance , DNA/metabolism , DNA/radiation effects , DNA Damage , DNA Repair , DNA, Bacterial/metabolism , DNA, Bacterial/radiation effects , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deinococcus/enzymology , Deinococcus/genetics , Exonucleases/chemistry , Gamma Rays , Histidinol-Phosphatase/genetics , Histidinol-Phosphatase/metabolism
20.
DNA Repair (Amst) ; 4(3): 359-66, 2005 Mar 02.
Article in English | MEDLINE | ID: mdl-15661659

ABSTRACT

Double labeling of interphase and metaphase chromosomes by 5-chlorodeoxyuridine (CldU) and 5-iododeoxyuridine (IdU) has been used in studies of the dynamics of DNA replication. Here, we have used this approach and confocal microscopy to analyze sites of DNA repair synthesis during nucleotide excision repair (NER) in quiescent human fibroblasts. Surprisingly, we have found that when both precursors are added at the same time to UV-irradiated cells they label different sites in the nucleus. In contrast, even very short periods of simultaneous IdU+CldU labeling of S-phase cells produced mostly overlapped IdU and CldU replication foci. The differential labeling of repair sites might be due to compartmentalization of I-dUTP and Cl-dUTP pools, or to differential utilization of these thymidine analogs by DNA polymerases delta and epsilon (Poldelta and Polepsilon). To explore the latter possibility we used purified mammalian polymerases to find that I-dUTP is efficiently utilized by both Poldelta and Polepsilon. However, we found that the UV-induced incorporation of IdU was more strongly stimulated by treatment of cells with hydroxyurea than was incorporation of CldU. This indicates that there may be distinct IdU and CldU-derived nucleotide pools differentially affected by inhibition of the ribonucleotide reductase pathway of dNTP synthesis and that is consistent with the view that differential incorporation of IdU and CldU during NER may be caused by compartmentalization of IdU- and CldU-derived nucleotide pools.


Subject(s)
DNA Repair , DNA/metabolism , Deoxyuridine/metabolism , Ultraviolet Rays , Cells, Cultured , DNA Polymerase II/metabolism , DNA Polymerase III/metabolism , Humans , Hydroxyurea/pharmacology , Microscopy, Confocal , S Phase
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