Coincident <i>In Vitro</i> Analysis of DNA‐PK‐Dependent and ‐Independent Nonhomologous End Joining

Hendrickson, Cynthia L.; Purkayastha, Shubhadeep; Pastwa, Elżbieta; Neumann, Ronald D.; Winters, Thomas A.

doi:10.4061/2010/823917

Cited by 6 publications

(4 citation statements)

References 86 publications

(103 reference statements)

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“…CI values at the ED 50 , ED 75 and ED 90 doses were 0.8854, 0.33208 and 0.18683, respectively, suggesting that DDRI‐18 and NU7026 have nearly additive to synergistic effects at this concentration range. Taken together, these results suggest that DDRI‐18 may have a different mode of action from NU7026 and may inhibit the DNA‐PK independent NHEJ DNA repair, which has been studied in several previous reports (Yano et al ., 2008; Hendrickson et al ., 2010). However, the specific target of the DDRI‐18 inhibitory mechanism remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Characterization of DDRI‐18 (3,3′‐(1H,3′H‐5,5′‐bibenzo[d]imidazole‐2,2′‐diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response

Jeong

Kim

et al. 2012

British J Pharmacology

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BACKGROUND AND PURPOSE Recently, the DNA damage response (DDR) has emerged as a promising target for anticancer drug development. In our previous study, we identified several DDR‐inhibiting compounds via high‐content screening of a small molecule library using γH2AX foci as a biomarker. Here, we studied the effects of the DNA damage response inhibitor DDRI‐18 (3,3′‐(1H,3′H‐5,5′‐bibenzo[d]imidazole‐2,2′‐diyl)dianiline) on DDR. EXPERIMENTAL APPROACH Osteosarcoma U2OS cells were treated with etoposide to induce DDR. The nuclear foci of γH2AX and other signalling molecules in DDR were visualized by immunofluorescence and quantified using an IN Cell Analyzer. The DNA repair capacity of cells was analysed using the comet assay and in vivo DNA end‐joining assay. Cell survival after drug treatment was quantified using the MTT assay, and apoptotic cell death was analysed by Annexin V staining and flow cytometry. KEY RESULTS DDRI‐18 inhibited the non‐homologous end‐joining (NHEJ) DNA repair process and delayed the resolution of DNA damage‐related proteins (γH2AX, ATM and BRCA1) from DNA lesions at a later phase of DDR. Furthermore, DDRI‐18 enhanced the cytotoxic effects of anticancer DNA‐damaging drugs, including etoposide, camptothecin, doxorubicin and bleomycin. This synergistic effect on cell death was shown to be due to caspase‐dependent apoptosis. CONCLUSIONS AND IMPLICATIONS We identified a chemical compound, DDRI‐18, that has chemosensitization activity. Although the target molecule and mechanism of action of DDRI‐18 remain unknown, DDRI‐18 is an effective chemosensitizing agent and may improve the therapy with classical anticancer drugs.

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Section: Discussionmentioning

confidence: 99%

Characterization of DDRI‐18 (3,3′‐(1H,3′H‐5,5′‐bibenzo[d]imidazole‐2,2′‐diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response

Jeong

Kim

et al. 2012

British J Pharmacology

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“…Notably, low Mg 2+ concentration were found to favor DNA-PKcs-dependent end-joining activity [51], whereas high Mg 2+ concentration (10 mM) facilitated DNA-PK-independent reaction [34]. In addition, A-EJ preferred high DNA ends/protein molar ratios [44,52] or was favored by volume excluders like PEG [53], possibly indicating a weak intrinsic synapsis activity at DNA ends.…”

Section: A-ej Toolsmentioning

confidence: 90%

Alternative end-joining pathway(s): Bricolage at DNA breaks

et al. 2014

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To cope with DNA double strand break (DSB) genotoxicity, cells have evolved two main repair pathways: homologous recombination which uses homologous DNA sequences as repair templates, and non-homologous Ku-dependent end-joining involving direct sealing of DSB ends by DNA ligase IV (Lig4). During the last two decades a third player most commonly named alternative end-joining (A-EJ) has emerged, which is defined as any Ku- or Lig4-independent end-joining process. A-EJ increasingly appears as a highly error-prone bricolage on DSBs and despite expanding exploration, it still escapes full characterization. In the present review, we discuss the mechanism and regulation of A-EJ as well as its biological relevance under physiological and pathological situations, with a particular emphasis on chromosomal instability and cancer. Whether or not it is a genuine DSB repair pathway, A-EJ is emerging as an important cellular process and understanding A-EJ will certainly be a major challenge for the coming years.

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“…The high concentration of KU and DNA-PKcs could potentiate relative lowaffinity interaction with structured RNA, including the telomerase RNA template, which might contribute to the prominent telomere abnormalities 119,120 in human cells lost KU or DNA-PKcs. Accordingly, while DNA-PKcs or KU null mice are viable, human cells, even cancer cells, cannot tolerate the complete and persistent loss of KU 120 or DNA-PKcs without adaptive changes of other genes, including reducing ATM protein levels 70,121 . Several human patients derived cell lines contain spontaneous mutations of DNA-PKcs are viable, but whether the residual DNA-PKcs protein or activity or adaptive changes are essential for their long-term culture is yet to be determined 54,56,122 .…”

Section: The Role Of Dna-pk and Its Kinase Activity In Cnhej And V(d)j Recombinationmentioning

confidence: 99%

The recent advances in non-homologous end-joining through the lens of lymphocyte development

2020

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Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocytespecific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Coincident In Vitro Analysis of DNA‐PK‐Dependent and ‐Independent Nonhomologous End Joining

Cited by 6 publications

References 86 publications

Characterization of DDRI‐18 (3,3′‐(1H,3′H‐5,5′‐bibenzo[d]imidazole‐2,2′‐diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response

Characterization of DDRI‐18 (3,3′‐(1H,3′H‐5,5′‐bibenzo[d]imidazole‐2,2′‐diyl)dianiline), a novel small molecule inhibitor modulating the DNA damage response

Alternative end-joining pathway(s): Bricolage at DNA breaks

The recent advances in non-homologous end-joining through the lens of lymphocyte development

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