Distinct Pathways of Nonhomologous End Joining That Are Differentially Regulated by DNA-dependent Protein Kinase-mediated Phosphorylation

Udayakumar, Durga; Bladen, Catherine L.; Hudson, Farlyn Z.; Dynan, William S.

doi:10.1074/jbc.m306470200

Cited by 57 publications

(54 citation statements)

References 36 publications

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“…Dynan and colleagues (20) and Iliakis and colleagues (37) used in vitro end joining systems to demonstrate that kinase inactive DNA-PKcs can inhibit DNA-PKcsindependent subpathway(s) of NHEJ. Additionally, several reports using cellular systems have shown that inactivating DNAPKcs (by kinase inhibitors) blocks other DNA repair and metabolism pathways.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas full-length DNA-PKcs efficiently complements both the coding joint and more modest signal joint deficit of the V3 cell line, neither isoform II nor III supported either coding or signal end joining. DNA-PKcs independent subpathway(s) of NHEJ have been reported (20). Residual signal end joining in DNA-PKcs deficient cells (dependent on Ku, XRCC4, and DNA ligase IV) may represent such a pathway.…”

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

confidence: 99%

“…17 and 18). An emerging consensus is that DNA-PK acts as a ''gatekeeper'' to regulate access of DNA ends to other repair factors like XRCC4, DNA ligase IV, and Artemis (19)(20)(21)(22).…”

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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: Discussionmentioning

confidence: 99%

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

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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“…Examination of a substrate containing histone H1 has provided the first demonstration of DNA-PK-dependent endjoining reconstituted by using purified or in-vitro-expressed proteins. In contrast, NHEJ activity by partially purified cell-free extracts has been shown to require DNA-PK activity in a reaction that uses naked DNA as a substrate (8,37), and DNA-PK has been shown to be required for LX-mediated ligation in the presence of a partially purified factor of Ϸ200 kDa (8). Interestingly, we have observed that addition of naked DNA to cell-free extracts prepared under conditions that support NHEJ results in the binding of histone H1 to DNA (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…Although DNA-PK phosphorylation is required for the hairpin cleavage activity of Artemis and the biochemical activities of the DNA-PK and LX complexes have been established in vitro, little is known about the role of DNA-PK in DSB rejoining or its in vivo phosphorylation targets. Current models suggest that DNA-PK may regulate NHEJ (4)(5)(6)(7)(8).…”

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confidence: 99%

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

Kysela

Chovanec

Jeggo

2005

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

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DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV͞XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.chromatin ͉ DNA double-stranded break repair ͉ nonhomologous end-joining D NA nonhomologous end-joining (NHEJ) is the major mechanism for the repair of DNA double-stranded (ds) breaks (DSBs) in mammalian cells and functions to effect DNA rearrangement during V(D)J recombination. Five proteins forming two complexes are required for NHEJ in mammalian cells, namely DNA ligase IV͞XRCC4 (LX) complex and the DNA-dependent protein kinase (DNA-PK) complex, which encompasses the two subunits of the Ku heterodimer and a large catalytic subunit (DNA-PKcs) (1, 2). Recently, a sixth protein, Artemis, has been shown to cleave the hairpin intermediate generated during V(D)J recombination (3). Although DNA-PK phosphorylation is required for the hairpin cleavage activity of Artemis and the biochemical activities of the DNA-PK and LX complexes have been established in vitro, little is known about the role of DNA-PK in DSB rejoining or its in vivo phosphorylation targets. Current models suggest that DNA-PK may regulate NHEJ (4-8).Recently, we have shown that LX is most efficient on substrates Ϸ400 bp long and inefficient on short (Ϸ50-bp) oligomers (9). Ku can stimulate LX ligation in a manner that requires freedom for Ku to translocate inwardly on the DNA (9-11). However, the presence of the intact DNA-PK complex does not provide any further stimulation of end-joining, and, indeed, its presence can be inhibitory (see Results). Our finding that efficient LX ligation in the presence of Ku requires inward translocation of Ku raises the question of how the presence of nucleosomes and the complex structural packaging of DNA into chromatin might impact the efficiency of NHEJ. The fundamental structural unit of chromatin is the nucleosome, an octameric complex of two copies of each core histone (H2A, H2B, H3, an...

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TheMre11 Nuclease Complex

Hopfner

Bemeleit

Lammens

2004

Handbook of Metalloproteins

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The repair of DNA double‐strand breaks (DSBs) is necessary to maintain genome stability and prevent cell aberration and cancer development. Meiotic recombination 11 (Mre11) and radiation sensitivity 50 (Rad50) form an evolutionary conserved complex (denoted Mre11 complex) that is a key factor in the detection, repair, and signaling of DSBs. The Mre11 complex possesses ATP‐stimulated DNA binding, DNA endo‐ and exonuclease activities as well as DNA cross‐linking functions. The Mre11 complex is a primary sensor for DNA DSBs and is involved in many aspects of the repair of and cellular response to DNA DSBs. Its activity is controlled by at least three different metal binding sites, a manganese‐dependent nuclease (Mre11 phosphodiesterase domain), a magnesium‐dependent ATPase (Rad50 ABC ATPase domain), and a zinc‐mediated DNA tethering function, located at the apex of a long Rad50 coiled‐coil domain. Although the mechanism remains to be shown, current structural and functional data indicate that the Mre11 complex is an ATP‐dependent cross‐linker and a processing factor for DNA ends.

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Distinct Pathways of Nonhomologous End Joining That Are Differentially Regulated by DNA-dependent Protein Kinase-mediated Phosphorylation

Cited by 57 publications

References 36 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)

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

TheMre11 Nuclease Complex

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