scholarly journals Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster.

2006 ◽  
Vol 53 (1) ◽  
pp. 233-236 ◽  
Author(s):  
Kamil Brzóska ◽  
Marcin Kruszewski ◽  
Irena Szumiel
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
In Vitro Test ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Dependent Protein ◽  
Phenotypic Features ◽  
Repair Defect

Cells with mutated autophosphorylation sites in the ABCDE cluster of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are defective in the repair of ionising radiation-induced DSB, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. Nevertheless, the mutated DNA-PK is able to undergo ATP-dependent autophosphorylation and inactivation. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in DSB repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line. The purpose of this work was to examine the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells. Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences coding for the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PK autophosphorylation sites (ABCDE casette).

scholarly journals Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1091
Author(s):  
Aya Kurosawa
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Strand Break ◽  
In Vivo Studies ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

scholarly journals Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair

2003 ◽  
Vol 23 (16) ◽  
pp. 5836-5848 ◽  
Author(s):  
Qi Ding ◽  
Yeturu V. R. Reddy ◽  
Wei Wang ◽  
Timothy Woods ◽  
Pauline Douglas ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Strand Break ◽  
Catalytic Subunit ◽  
Dna Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Major Cluster ◽  
Dependent Protein

ABSTRACT The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.

DNA end sequestration by DNA-dependent protein kinase and end joining of sterically constrained substrates in whole-cell extracts

2003 ◽  
Vol 42 (4) ◽  
pp. 279-287 ◽  
Author(s):  
Jae Wan Lee ◽  
Kedar V. Inamdar ◽  
Michele F. Hannah ◽  
Susan P. Lees-Miller ◽  
Lawrence F. Povirk
Keyword(s):  
Protein Kinase ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dependent Protein
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