ABSTRACT
Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as 'gaps' in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.
Subject(s)
Actins , Stereocilia , Animals , Mice , Actin Cytoskeleton/metabolism , Actins/metabolism , Hair Cells, Auditory/metabolism , Hair Cells, Auditory, Inner/metabolism , Stereocilia/metabolismABSTRACT
The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.
Subject(s)
Acid Anhydride Hydrolases/metabolism , Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , MRE11 Homologue Protein/metabolism , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , RecQ Helicases/metabolism , Acid Anhydride Hydrolases/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , MRE11 Homologue Protein/genetics , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , RecQ Helicases/geneticsABSTRACT
Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.
Subject(s)
DNA Replication , RecQ Helicases/metabolism , Replication Origin , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Origin Recognition Complex/metabolism , Peptide Initiation Factors/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RecQ Helicases/genetics , S Phase , Transcriptional ActivationABSTRACT
RecQL4 has been shown to be involved in DNA replication and repair, but its role in DNA damage checkpoint pathway has not been reported. Here, we show that RecQL4 plays an important role in the activation of ataxia telangiectasia mutated (ATM)-dependent checkpoint pathway in human cells. Cells depleted with RecQL4 or Rothmund-Thomson syndrome cells showed significant impairment in the activation of ATM and the downstream effector proteins such as checkpoint kinase 2 and p53 after DNA damage. This defect was recovered with the expression of wild type RecQL4 but not any mutant RecQL4 proteins with defective helicase activities. While RecQL4 failed to show any direct interaction with ATM, it stably interacted with the Mre11-Rad50-Nbs1 complex that is essential for the activation of ATM and was localized on the DNA damage foci. Thus, our results suggest that the helicase activity of RecQL4 plays an important role in the activation of ATM-dependent checkpoint pathway against DNA double strand breaks in human cells.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , DNA Repair , DNA/genetics , RecQ Helicases/genetics , Rothmund-Thomson Syndrome/genetics , Acid Anhydride Hydrolases , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genetic Complementation Test , Humans , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Protein Binding , RecQ Helicases/deficiency , Rothmund-Thomson Syndrome/metabolism , Rothmund-Thomson Syndrome/pathology , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Cell Cycle Proteins/genetics , DNA End-Joining Repair , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Nuclear Proteins/genetics , Trans-Activators/genetics , Adaptor Proteins, Signal Transducing , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Transformed , Cell Line, Tumor , Chromatin/chemistry , Chromatin/metabolism , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded , DNA, Neoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Deletion , HEK293 Cells , HeLa Cells , Histones/genetics , Histones/metabolism , Humans , Molecular Chaperones , Nuclear Proteins/metabolism , Phosphorylation , Signal Transduction , Trans-Activators/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of MCM8-9 are required for recruitment of MRE11 to foci of DNA damage. Homozygous deletion of the MCM9 found in various cancers sensitizes a cancer cell line to interstrand-crosslinking (ICL) agents. A cancer-derived point mutation or an SNP on MCM8 associated with premature ovarian failure (POF) diminishes the functional activity of MCM8. Therefore, the MCM8-9 complex facilitates DNA resection by the MRN complex during HR repair, genetic or epigenetic inactivation of MCM8 or MCM9 are seen in human cancers, and genetic inactivation of MCM8 may be the basis of a POF syndrome.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Minichromosome Maintenance Proteins/metabolism , Nuclear Proteins/metabolism , ATP-Binding Cassette Transporters/metabolism , Acid Anhydride Hydrolases , Animals , Carrier Proteins/metabolism , Chromatin Immunoprecipitation , DNA Damage , DNA Repair , DNA, Single-Stranded , HEK293 Cells , HeLa Cells , Humans , MRE11 Homologue Protein , Mice , Recombinational DNA Repair , Tumor Suppressor Proteins/metabolism , Xenopus , Xenopus Proteins/metabolismABSTRACT
Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells.
Subject(s)
DNA-Binding Proteins/metabolism , Minichromosome Maintenance Proteins/metabolism , RecQ Helicases/physiology , Replication Origin , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Damage , DNA Replication , HeLa Cells , Humans , Protein Serine-Threonine Kinases/metabolismABSTRACT
Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1(ßTRCP)-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.
Subject(s)
Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Gene Expression Regulation, Neoplastic , Replication Origin/genetics , Ubiquitin-Protein Ligases/metabolism , Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cells/drug effects , DNA Replication/genetics , Doxorubicin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Gene Knockdown Techniques , HeLa Cells , Histones/metabolism , Humans , Molecular Chaperones , RNA Polymerase II/metabolismABSTRACT
Though the G(1) checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G(1) phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G(1) phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G(1) arrest in various other checkpoint pathways.
Subject(s)
Cell Cycle/drug effects , Chromatin/metabolism , DNA Replication/drug effects , DNA-Binding Proteins/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mimosine/pharmacology , Cell Cycle/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Chromatin/genetics , DNA Replication/genetics , DNA-Binding Proteins/genetics , G1 Phase/drug effects , G1 Phase/genetics , HeLa Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Protein Binding/drug effects , Protein Binding/geneticsABSTRACT
PER3 is a member of the PERIOD genes, but does not play essential roles in the circadian clock. Depletion of Per3 by siRNA almost completely abolished activation of checkpoint kinase 2 (Chk2) after inducing DNA damage in human cells. In addition, Per3 physically interacted with ATM and Chk2. Per3 overexpression induced Chk2 activation in the absence of exogenous DNA damage, and this activation depended on ATM. Per3 overexpression also led to the inhibition of cell proliferation and apoptotic cell death. These combined results suggest that Per3 is a checkpoint protein that plays important roles in checkpoint activation, cell proliferation and apoptosis.
Subject(s)
Circadian Clocks/genetics , Period Circadian Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , Apoptosis/genetics , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Proliferation , Checkpoint Kinase 2 , DNA Damage/genetics , DNA-Binding Proteins/metabolism , Down-Regulation/genetics , Enzyme Activation/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Period Circadian Proteins/deficiency , Period Circadian Proteins/metabolism , RNA, Small Interfering/genetics , Signal Transduction/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G(1)/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , RecQ Helicases/chemistry , RecQ Helicases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Cell Cycle , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Minichromosome Maintenance Complex Component 2 , Minichromosome Maintenance Complex Component 7 , Minichromosome Maintenance Proteins , Multiprotein Complexes , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/metabolism , RecQ Helicases/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.
Subject(s)
Apoptosis , Cell Cycle Proteins/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Protein Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , HeLa Cells , Humans , MAP Kinase Kinase 4/metabolism , Models, Biological , Signal Transduction , Time FactorsABSTRACT
Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated beta-galactosidase (SA-beta-gal), which is defined as beta-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-beta-gal and its cellular roles in senescence are not known. We demonstrate here that SA-beta-gal activity is expressed from GLB1, the gene encoding lysosomal beta-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive G(M1)-gangliosidosis, which have defective lysosomal beta-galactosidase, did not express SA-beta-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-beta-gal. GLB1 mRNA depletion also prevented expression of SA-beta-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-beta-gal induction during senescence was due at least in part to increased expression of the lysosomal beta-galactosidase protein. These results also indicate that SA-beta-gal is not required for senescence.
Subject(s)
Cellular Senescence , Lysosomes/enzymology , beta-Galactosidase/metabolism , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/pathology , Gangliosidoses/enzymology , HeLa Cells , Humans , Mutation/genetics , RNA Interference , beta-Galactosidase/geneticsABSTRACT
The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53-Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated beta-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells.