scholarly journals The Absence of the DNA-Dependent Protein Kinase Catalytic Subunit in Mice Results in Anaphase Bridges and in Increased Telomeric Fusions with Normal Telomere Length and G-Strand Overhang

2001 ◽  
Vol 21 (11) ◽  
pp. 3642-3651 ◽  
Author(s):  
Fermı́n A. Goytisolo ◽  
Enrique Samper ◽  
Scott Edmonson ◽  
Guillermo E. Taccioli ◽  
Marı́a A. Blasco
Keyword(s):  
Protein Kinase ◽  
Telomere Length ◽  
Mammalian Cells ◽  
Catalytic Subunit ◽  
Dna Double Strand Breaks ◽  
Dependent Protein Kinase ◽  
Major Pathway ◽  
Anaphase Bridges ◽  
Dependent Protein ◽  
Ku Proteins

ABSTRACT The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs−/− cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.

scholarly journals Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions

2004 ◽  
Vol 381 (1) ◽  
pp. 165-174 ◽  
Author(s):  
Ramiro DIP ◽  
Hanspeter NAEGELI
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Strand Break ◽  
Holliday Junctions ◽  
Catalytic Subunit ◽  
Cross Linking ◽  
Dependent Protein Kinase ◽  
Functional Link ◽  
Dependent Protein ◽  
Ku Proteins

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PKCS (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PKCS associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin–streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PKCS with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PKCS binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PKCS adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PKCS to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

scholarly journals Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates γ-H2AX

2010 ◽  
Vol 30 (6) ◽  
pp. 1368-1381 ◽  
Author(s):  
Pauline Douglas ◽  
Jianing Zhong ◽  
Ruiqiong Ye ◽  
Greg B. G. Moorhead ◽  
Xingzhi Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dna Double Strand Breaks ◽  
Dependent Protein Kinase ◽  
Histone H2ax ◽  
Dependent Protein

ABSTRACT The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G2/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (γ-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of γ-H2AX, the dissolution of IR-induced foci, and release from the G2/M checkpoint in vivo.

scholarly journals DNA-Dependent Protein Kinase Catalytic Subunit: The Sensor for DNA Double-Strand Breaks Structurally and Functionally Related to Ataxia Telangiectasia Mutated

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1143
Author(s):  
Yoshihisa Matsumoto ◽  
Anie Day D. C. Asa ◽  
Chaity Modak ◽  
Mikio Shimada
Keyword(s):  
Protein Kinase ◽  
Ataxia Telangiectasia ◽  
Catalytic Subunit ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is composed of a DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/Ku80 heterodimer. DNA-PK is thought to act as the “sensor” for DNA double-stranded breaks (DSB), which are considered the most deleterious type of DNA damage. In particular, DNA-PKcs and Ku are shown to be essential for DSB repair through nonhomologous end joining (NHEJ). The phenotypes of animals and human individuals with defective DNA-PKcs or Ku functions indicate their essential roles in these developments, especially in neuronal and immune systems. DNA-PKcs are structurally related to Ataxia–telangiectasia mutated (ATM), which is also implicated in the cellular responses to DSBs. DNA-PKcs and ATM constitute the phosphatidylinositol 3-kinase-like kinases (PIKKs) family with several other molecules. Here, we review the accumulated knowledge on the functions of DNA-PKcs, mainly based on the phenotypes of DNA-PKcs-deficient cells in animals and human individuals, and also discuss its relationship with ATM in the maintenance of genomic stability.

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