Ku Stimulation of DNA Ligase IV-dependent Ligation Requires Inward Movement along the DNA Molecule

Kysela, Boris; Doherty, Aidan J.; Chovanec, Miroslav; Stiff, Thomas; Ameer-Beg, Simon; Vojnovic, B.; Girard, Pierre‐Marie; Jeggo, Penny A.

doi:10.1074/jbc.m303273200

Cited by 71 publications

(65 citation statements)

References 28 publications

(39 reference statements)

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“…In support of this we have observed that adenylation activity is only detectable after treatment with PPi suggesting that the majority of the DNA ligase IV/Xrcc4 complexes expressed in insect cells are pre-adenylated [22] (data not shown). Additionally, we have found that non-adenylatable DNA ligase IV complexes are expressed at low levels in insect cells.…”

Section: Mutational Changes Within Motif Va Impact Upon Dna Ligase IVsupporting

confidence: 60%

“…[20,22] Human Xrcc4 was obtained from pCIneo-Xrcc4 by digestion with EcoRI and NotI, and inserted into the multiple cloning site of pFasBac Dual vector (Invitrogen) generating pFastBacDual-Xrcc4.…”

Section: Methodsmentioning

confidence: 99%

“…It has been shown previously that WT DNA ligase IV/Xrcc4 complex is able to bind efficiently to a DNA fragment of 54bp length [22].…”

Section: Motif Va Is Dispensable For Dna Bindingmentioning

confidence: 96%

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Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

et al. 2006

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Penny (2006) Identification of a novel motif in DNA ligases exemplified by DNA ligase IV. DNA Repair, 5 (7). pp. 788-798. ISSN 1568-7864 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/17805/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the URL above for details on accessing the published version. Copyright and reuse:Sussex Research Online is a digital repository of the research output of the University.Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available.Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly however, such mutational changes do impact upon double stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggests that motif Va functions as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.3

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Section: Mutational Changes Within Motif Va Impact Upon Dna Ligase IVsupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

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Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

et al. 2006

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“…The Ku heterodimer (orange and red) binds DNA ends exposed by a double-strand break, recruits DNA-PKcs and forms the catalytically active protein kinase DNA-PK. DNA Ligase IV is recruited as part of a complex with XRCC4 and XLF and requires inward translocation of Ku to permit binding of DNA Ligase IV complex [17]. End-ligation is catalyzed by DNA Ligase IV.…”

Section: Resultsmentioning

confidence: 99%

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

Hanakahi

2007

Protein Expression and Purification

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The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic E. coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (~9000Å 2 [1]), which suggests that herterodimerization may be important for protein stability. N-terminally his 6 -tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of his 6 -tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli. KeywordsKu; DNA-PK; non-homologous end-joining; NHEJ The repair of DNA double strand breaks (DSB) is critical to the maintenance of genomic integrity. The importance of DSB repair is highlighted by the fact that unrepaired DSBs can result in the loss of genetic information, chromosomal translocation and even cell death [2]. An important mechanism for DSB repair is non-homologous end-joining (NHEJ) -a homology independent process that is used for the repair DSBs during G0, G1 and early-S phases of the cell cycle [2]. As most of the cells in the mammalian body are terminally differentiated (G0), NHEJ is the major DSB repair pathway used by mammals.Thus far, six mammalian NHEJ proteins have been described. Central to the DSB repair process is DNA Ligase IV and the proteins XRCC4 and XLF, which are though to function as a complex in the ligation reaction [3][4][5][6] (Fig. 1C). Three other proteins -the Ku heterodimer ( Fig. 1) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) -comprise the DNAdependent protein kinase (DNA-PK) [7]. Studies employing mouse models have demonstrated that genomic stability depends upon the fidelity of DSB repair through the NHEJ pathway and deficiencies in NHEJ have been linked to cancer [2,8].Correspondence to: Les A. Hanakahi. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The DNA end-binding protein Ku protein plays an important role in mammalian NHEJ. Owing to its ring-shaped structure,...

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“…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).…”

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confidence: 99%

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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Ku Stimulation of DNA Ligase IV-dependent Ligation Requires Inward Movement along the DNA Molecule

Cited by 71 publications

References 28 publications

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

2-Step purification of the Ku DNA repair protein expressed in Escherichia coli

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

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