Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination

Kienker, Laura J.; Shin, Euy Kyun; Meek, Katheryn

doi:10.1093/nar/28.14.2752

Cited by 93 publications

(99 citation statements)

References 57 publications

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“…3B). Consistent with previous reports (14,15), complementation of the radiosensitivity of murine SCID cells was only observed in transfectants expressing the kinase competent form of DNA-PKcs (Fig. 3C).…”

Section: Dna-pkcs Isoforms II and Iii Do Not Complement The Nhej Defisupporting

confidence: 92%

“…This exon encodes a 31-aa insertion at position 3797 within the PI3-kinase domain of DNA-PKcs. Transcript I (which includes exon 80, referred to hereafter as full-length or WT DNA-PKcs) is the predominant form expressed in cell lines, and this form encodes an active protein kinase that is fully functional in NHEJ (14,15). The second major splice variant (transcript II) retains intron D (isoform II) inserting an additional 67 nt into the DNA-PKcs transcript.…”

Section: Splicementioning

confidence: 99%

“…Because DNA-PK's protein kinase activity is required for its functional role in NHEJ, it has been suggested that DNA-PK may activate downstream repair mediators (14,15). To date, the only physiologically relevant DNA-PK target defined is DNA-PK itself (16).…”

mentioning

confidence: 99%

“…The two splice variants are only weakly expressed in cultured cell lines; however, expression of the splice variants was similar to full-length DNA-PKcs in all normal human tissues examined. Transfection studies demonstrate (14,15) that the full-length DNA-PKcs transcript generates an active form of the protein, which fully complements the DNA repair defects in DNA-PKcs-deficient cell lines. In this article, we demonstrate that the two alternative isoforms lack protein kinase enzymatic activity and thus cannot function in NHEJ.…”

mentioning

confidence: 99%

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Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Convery

Shin²,

Ding

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G0͞G1 (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G2. How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNAPKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.DNA repair ͉ DNA-dependent protein kinase ͉ nonhomologous DNA end joining pathway

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Section: Dna-pkcs Isoforms II and Iii Do Not Complement The Nhej Defisupporting

confidence: 92%

Section: Splicementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Convery

Shin²,

Ding

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Classical inhibitors The fact that DNA-PK kinase activity is essential for its function in both DNA DSB repair and V(D)J recombination (Kurimasa et al, 1999;Beamish et al, 2000;Kienker et al, 2000) opens the door for the development of drugs that can specifically inhibit this vital kinase activity. Initial reports described small-molecule inhibition of DNA-PK using two nonselective PI-3 kinase inhibitors: wortmannin and LY294002.…”

Section: Small-molecule Inhibitors Of Dna-pkmentioning

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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Phosphorylation of Artemis following irradiation‐induced DNA damage

et al. 2004

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Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin-sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR-induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR-induced phosphorylation is mainly, although not solely, dependent on Ataxia-telangiectasia-mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro-generated Artemis mutants, which present impaired IR-induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis-deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA-PKcs and ATR depending upon the type of DNA damage.

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Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination

Cited by 93 publications

References 57 publications

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

The life and death of DNA-PK

Phosphorylation of Artemis following irradiation‐induced DNA damage

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