scholarly journals DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

2020 ◽  
Vol 6 (2) ◽  
pp. eaay0922 ◽  
Author(s):  
Rajashree A. Deshpande ◽  
Logan R. Myler ◽  
Michael M. Soniat ◽  
Nodar Makharashvili ◽  
Linda Lee ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Mrn Complex ◽  
Dependent Protein ◽  
Dna Ends

The repair of DNA double-strand breaks occurs through nonhomologous end joining or homologous recombination in vertebrate cells—a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here, we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK–bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from nonhomologous end joining to homologous recombination by facilitating DNA-PK removal.

scholarly journals DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

2018 ◽  
Author(s):  
Rajashree A. Deshpande ◽  
Logan R. Myler ◽  
Michael M. Soniat ◽  
Nodar Makharashvili ◽  
Linda Lee ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Homologous Recombination ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Mrn Complex ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Dna Ends

AbstractThe repair of DNA double-strand breaks occurs through non-homologous end joining or homologous recombination in vertebrate cells - a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from non-homologous end joining to homologous recombination by facilitating DNA-PK removal.One Sentence SummaryDNA-dependent protein kinase, an enzyme critical for non-homologous repair of DNA double-strand breaks, also stimulates end processing for homologous recombination.

scholarly journals DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?

2007 ◽  
Vol 179 (2) ◽  
pp. 183-186 ◽  
Author(s):  
Eric Weterings ◽  
David J. Chen
Keyword(s):  
Protein Kinase ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Dsb Repair ◽  
Dependent Protein Kinase ◽  
Dna Molecule ◽  
Dna Double Strand Break ◽  
Dependent Protein ◽  
Multiple Keys

The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.

scholarly journals Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation

2020 ◽  
Vol 48 (16) ◽  
pp. 9098-9108 ◽  
Author(s):  
Katheryn Meek
Keyword(s):  
Protein Kinase ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Stepwise Mechanism ◽  
Dependent Protein ◽  
Enzymatic Activation ◽  
Non Homologous End Joining ◽  
Dna Ends ◽  
Step Mechanism

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

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.

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