DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination

Yu, Yaping; Wang, Wei; Ding, Qi; Ye, Ruiqiong; Chen, Dawn; Merkle, Dennis; Schriemer, David C.; Meek, Katheryn; Lees‐Miller, Susan P.

doi:10.1016/s1568-7864(03)00143-5

Cited by 109 publications

(127 citation statements)

References 38 publications

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“…Residue Ser-260 of XRCC4 was clearly phosphorylated, which is consistent with previous reports (53,54). An additional site of human XRCC4, either Ser-313 or Ser-318, was also phosphorylated, again consistent with a recent analysis of XRCC4 phosphorylation data (54). The additional minor DNA-PK phosphorylation sites that were identified in XRCC4 (54) were not found in this study as 32 P labeling before mass spectrometry was not utilized in our method.…”

Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 92%

“…The Lig4 and XRCC4 bands were excised and analyzed by mass spectrometry. Residue Ser-260 of XRCC4 was clearly phosphorylated, which is consistent with previous reports (53,54). An additional site of human XRCC4, either Ser-313 or Ser-318, was also phosphorylated, again consistent with a recent analysis of XRCC4 phosphorylation data (54).…”

Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 91%

“…An additional site of human XRCC4, either Ser-313 or Ser-318, was also phosphorylated, again consistent with a recent analysis of XRCC4 phosphorylation data (54). The additional minor DNA-PK phosphorylation sites that were identified in XRCC4 (54) were not found in this study as 32 P labeling before mass spectrometry was not utilized in our method. One phosphorylation residue of human Lig4 was identified as Thr-650 (Fig.…”

Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 91%

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Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase

Wang

Nnakwe

Lane

et al. 2004

Journal of Biological Chemistry

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DNA ligase IV (Lig4), x-ray cross-complementation group 4 (XRCC4), and DNA-dependent protein kinase (DNA-PK) are essential mammalian nonhomologous end joining proteins used for V(D)J recombination and DNA repair. Previously a Lig4 peptide was reported to be an in vitro substrate for DNA-PK, but the phosphorylation state of Lig4 protein in vivo is not known. In this study, we report that a full-length Lig4 construct was expressed as a phosphoprotein in the cell. Also the fulllength Lig4 protein, in complex with XRCC4, was an in vitro substrate for DNA-PK. Using tandem mass spectrometry, we identified a DNA-PK phosphorylation site at Thr-650 in human Lig4 and a potential second phosphorylation site at Ser-668 or Ser-672. Phosphorylation of Lig4 per se was not required for Lig4 DNA end joining activity. Substitution of these amino acids with alanine, individually or in combination, led to changes in Lig4 protein stability of mouse Lig4. The phosphomimetic mutation S650D returned Lig4 stability to that of the wild-type protein. Furthermore DNA-PK was found to negatively regulate Lig4 protein stability. Our results suggest that Lig4 stability is regulated by multiple factors, including interaction with XRCC4, phosphorylation status, and possibly Lig4 conformation.Nonhomologous end joining (NHEJ) 1 is an efficient mechanism used by mammalian cells to repair DNA double strand breaks (1, 2) and is also required for the process of V(D)J recombination, the rearrangement of immunoglobulin and Tcell receptor genes essential for the generation of a diverse immune response (3). XRCC4 and DNA ligase IV (Lig4), which form a ligation complex in the cell (4, 5), are two critical proteins involved in these two processes (6 -8).The five most well studied proteins that are required for both NHEJ and V(D)J recombination in mammalian cells are Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNAPKcs), XRCC4, and Lig4. The heterodimeric Ku protein, comprising subunits Ku70 and Ku80, binds strongly to doublestranded DNA ends (9). When bound to DNA, Ku recruits DNA-PKcs and activates its kinase activity (10, 11). The XRCC4⅐Lig4 protein complex is recruited to the DNA ends to complete ligation (4,5,12). An additional protein, Artemis, which has nuclease activity and can open DNA hairpin intermediates with DNA-PKcs during V(D)J recombination (13), has also been shown to be involved in both NHEJ and V(D)J recombination (14, 15).Mice lacking NHEJ components have abnormal lymphocyte development due to defective V(D)J recombination (16). Mice deficient in Ku70, Ku80, XRCC4, or Lig4 also exhibit growth defects, premature senescence, and hypersensitivity to ionizing irradiation. Deficiency of XRCC4 or Lig4 is most severe, resulting in significant neuronal apoptosis and embryonic lethality (7,8,17). Embryonic fibroblast cells that are deficient for either DNA-PKcs or Artemis also exhibit cell type-specific ionizing irradiation hypersensitivity (16). Defects in the NHEJ factors have also been implicated in the generation of chromosome i...

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Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 92%

Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 91%

Section: Lig4 Is a Phosphoprotein And Potential In Vivosupporting

confidence: 91%

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Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase

Wang

Nnakwe

Lane

et al. 2004

Journal of Biological Chemistry

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“…Severe combined immunodeficient (SCID) mice with a defective DNA-PK cs gene Yu et al (2003) possess a premature aging phenotype and a defective V(D)J recombination phenotype, which results in an accumulation of unprocessed coding intermediates (Bogue et al, 1998). It has further been suggested that DNA-PK cs -deficient cells and DNA-PK cs À/À mice may also have some defect in signal joint formation (Jhappan et al, 1997;Gao et al, 1998;Taccioli et al, 1998).…”

Section: Dna-pk Defective Cellsmentioning

confidence: 99%

“…DNA-PK has been shown to phosphorylate serine 53 of XRCC4, but this event is dispensable for proficient V(D)J recombination (Mizuta et al, 1997). Yu et al (2003) identified XRCC4 serines 260 and 318 as major sites of DNA-PK phosphorylation and possibly serines 193, 259, 302, 313, 325 and 326 as well as threonine 321 as minor sites. However, an XRCC4 mutant with all nine DNA-PK phosphorylation sites mutated showed the same sensitivity to IR as those expressing the wildtype DNA-PK protein.…”

Section: Dna-pk Interactions Within the Nhej Machinarymentioning

confidence: 99%

The life and death of DNA-PK

et al. 2004

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Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous endjoining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK cs ), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK cs ), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK À/À mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR. The double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions, which if unrepaired severely threatens not only the integrity of the genome but also survival of the organism (Hoeijmakers, 2001;van Gent et al., 2001;Vilenchik and Knudson, 2003). DSBs can arise endogenously by cellular processes such as cleavage during immunoglobulin gene rearrangement (V(D)J recombination) and meiotic recombination. DSBs are also produced by exposure to ionizing radiation (IR), radiomimetic drugs, such as bleomycin, and the collapse of replication forks when the replication machinery encounters singlestranded breaks (SSBs) (Haber, 2000;Karran, 2000;Norbury and Hickson, 2001;Bassing et al., 2002). Unrepaired DSBs can activate cell cycle checkpoint arrests and signal for cell death (Jackson, 2001;Norbury and Hickson, 2001). Possibly even more detrimental to the cell are the unrepaired or misrepaired DSBs that lead to genomic rearrangements which ultimately destabilize the genome, a phenotype observed in a large number of malignancies (Lengauer et al., 1998;Hoeijmakers, 2001;Elliott and Jasin, 2002;Thompson and Schild, 2002;Shiloh, 2003;Vilenchik and Knudson, 2003). To complicate matters further, the location of the DSBs and the structure of the damaged DNA ends can vary depending on the damaging agent....

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X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

Hammel

Tainer

2021

Protein Science

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Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIVBRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.

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DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination

Cited by 109 publications

References 38 publications

Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase

Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase

The life and death of DNA-PK

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

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