ABSTRACT
ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while approximately 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.
Subject(s)
Cell Cycle Proteins/physiology , Cell Division/physiology , DNA Damage , DNA Repair , DNA-Binding Proteins/physiology , G2 Phase/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/physiology , Tumor Suppressor Proteins/physiology , Adaptor Proteins, Signal Transducing , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Division/radiation effects , Cells, Cultured , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Chromosomal Proteins, Non-Histone , DNA-Activated Protein Kinase/genetics , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endonucleases , Fibroblasts/cytology , Fibroblasts/physiology , G2 Phase/radiation effects , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction/physiology , Signal Transduction/radiation effects , Telomerase/genetics , Telomerase/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor p53-Binding Protein 1ABSTRACT
In mammalian cells, non-homologous end joining (NHEJ) is the major double strand break (DSB) repair mechanism during the G(1) phase of the cell cycle. It also contributes to DSB repair during the S and G(2) phases. Ku heterodimer, DNA PKcs, XRCC4 and DNA Ligase IV constitute the core NHEJ machinery, which joins directly ligatable ends. XRCC4-like factor/Cernunnos (XLF/Cer) is a recently discovered interaction partner of XRCC4. Current evidence suggests the following model for the role of XLF/Cer in NHEJ: after DSB induction, the XRCC4-DNA Ligase IV complex promotes efficient accumulation of XLF/Cer at DNA damage sites via constitutive interaction of the XRCC4 and XLF/Cer head domains and dependent on components of the DNA PK complex. Ku alone can stabilise the association of XLF/Cer with DNA ends. XLF/Cer stimulates ligation of complementary and non-complementary DNA ends by XRCC4-DNA Ligase IV. This activity involves the carboxy-terminal DNA binding region of XLF/Cer and could occur via different, non-exclusive modes: (i) enhancement of the stability of the XRCC4-DNA Ligase IV complex on DNA ends by XLF/Cer, (ii) modulation of the efficiency and/or specificity of DNA Ligase IV by binding of XLF/Cer to the XRCC4-DNA Ligase IV complex, (iii) promotion of the alignment of blunt or other non-complementary DNA ends by XLF/Cer for ligation. XLF/Cer promotes the preservation of 3' overhangs, restricts nucleotide loss and thereby promotes accuracy of DSB joining by XRCC4-DNA Ligase IV during NHEJ and V(D)J recombination.
Subject(s)
DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Humans , Protein Binding , Recombination, GeneticABSTRACT
Cells, which lacked the activity of the nuclease Artemis, retained approximately 10% of unrepaired double strand breaks (DSBs) at later timepoints after ionizing radiation. Ionizing radiation induced hyperphosphorylation of Artemis mainly by ATM and in ATM deficient cells to a minor extent by DNA PK. After induction of DSBs with modified ends by a high dose of calicheamicin gamma1, Artemis was phosphorylated by DNA PK. The type of calicheamicin gamma1-induced DSBs is likely to represent a subclass of DSBs induced by ionizing radiation. DNA PK-dependent phosphorylation of Artemis after treatment with DSB inducing agents increased the cellular retention of Artemis, maintained its interaction with DNA ends and activated its endonucleolytic activity. The following model is suggested: ATM-dependent phosphorylation of Artemis after ionizing radiation could prevent DNA PK-dependent phosphorylation and activation of undesired endonucleolytic activity at DSBs, which do not require endonucleolytic processing by Artemis. The Artemis:DNA PK complex could be involved in the repair of DSBs, which carry modified ends and are refractory to repair by otherwise lesion specific enzymes because of the presence of an inhibitory lesion in the opposite strand.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair/physiology , Nuclear Proteins/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Endonucleases , Humans , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
Subject(s)
DNA Damage , Histones/metabolism , Nuclear Proteins/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Line , Cells, Cultured , DNA Repair , DNA Repair Enzymes , DNA, Complementary/metabolism , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Radiation , Endonucleases , Epistasis, Genetic , Gamma Rays , Genetic Complementation Test , Humans , Infrared Rays , Intracellular Signaling Peptides and Proteins/metabolism , MRE11 Homologue Protein , Mice , Nuclear Proteins/metabolism , Phenotype , Phosphoproteins/metabolism , Phosphorylation , Severe Combined Immunodeficiency , Signal Transduction , Time Factors , Tumor Suppressor Proteins , Tumor Suppressor p53-Binding Protein 1 , X-RaysABSTRACT
In response to replication block or DNA damage in S phase the DNA replication and DNA damage checkpoints are activated. The current model in human predicts, that a Rad17/Replication factor C (RF-C) complex might serve as a recruitment complex for the Rad9/Hus1/Rad1 complex to sites of replication block or DNA damage. In this study we have investigated the fate of the Rad17/RF-C complex after treatment of synchronized Hela cells with the replication inhibitor hydroxyurea. In hydroxyurea treated cells the RF-C p37 subunit became more resistant to extraction. Moreover, co-immunoprecipitation studies with extracts of hydroxyurea treated cells showed an interaction of RF-C p37 with Rad17 and of PCNA with Rad9 and RF-C p37. An enhanced colocalization of Rad17 and PCNA in late S phase after hydroxyurea treatment was observed. Our data suggested, that upon replication block a Rad17/RF-C complex is recruited to sites of DNA lesions in late S phase, binds the Rad9/Hus1/Rad1 complex and enables it to interact with PCNA. An interaction of Rad17/RF-C with PCNA appears to be mediated by the small RF-C p37 subunit, suggesting that PCNA might provide communication between replication checkpoint control and DNA replication and repair.
