Stages, scaffolds and strings in the spatial organisation of non-homologous end joining: Insights from X-ray diffraction and Cryo-EM

Liang, Shikang; Chaplin, Amanda K.; Stavridi, Antonia Kefala; Appleby, Robert; Hnı́zda, Aleš; Blundell, Tom L.

doi:10.1016/j.pbiomolbio.2020.11.008

Cited by 10 publications

(5 citation statements)

References 135 publications

(232 reference statements)

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“…6), structure-guided drug discovery can exploit other specific pockets of DNA-PKcs in the development of allosteric inhibitors or proteolysis-targeting chimeric (PROTAC) drugs with enhanced potency and selectivity 31,32 . Degradation of DNA-PKcs eliminates not only its kinase activity but also the stage provided for downstream NHEJ and DNA damage response signalling 13,32,33 . However, PROTAC drugs cannot be guaranteed to be beneficial as previous studies have shown that inhibiting kinase activity has more potent effects than knockdown or knockout 34 .…”

Section: Articlementioning

confidence: 99%

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Liang

Thomas

Chaplin

et al. 2022

Nature

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The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

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Section: Articlementioning

confidence: 99%

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Liang

Thomas

Chaplin

et al. 2022

Nature

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“…These authors also added Ku protein which is known to promote weak and transient interaction between DNA ends with rather short dwell time (Ramsden & Gellert, 1998). A pair of proteins XRCC4 and Lig4 are capable to form complex between two XRCC4 disordered tails and two Lig4 molecules attached to two tails of XRCC4 dimer (Liang et al, 2020;Wu, 2019). Each one of the two Lig4 molecules is capable to embrace tightly only one DNA duplex.…”

Section: Dna Repair Systemsmentioning

confidence: 99%

Chromosome instability induced by mutations in TAD anchors leads to tumors

Luchnik

2021

GENOME INSTAB. DIS.

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In the light of recent data, a way is described between mutations in TAD anchors and stimulation of tumor growth driven by chromosome instability. At the end of this way every tumor sacrifices by a portion of its cells, dying necrotically, to stimulate remaining cells to proliferate. Necrosis (cell puncture and leakage of chemokine CXCL12) launches the mechanism of wound healing that stimulates cells to divide. Necrosis in cancer cells is induced by chromosome aberrations hindering anaphase cleavage furrow ingression and leading to membrane rupture. Inherited chromosome instability arises, because some of DNA loops (gene blocks, also called TADs) are torn off chromatid axes at their attachment points (anchors) because of mutations at some of these points. Namely, CTCF target sites (CCXXXAGG GGG ) at the basements of TAD loops acquire mutations in central Adenine and in 3-5 nucleotides just beyond the borders of this 11 bp sequence. The sequence itself is degenerate between different TADs mainly at the X nucleotides, rarely at some others. Interstitial satellite DNA (SAT) adjacent to TADs anchors begins to behave such as telomeric satellite DNA. This launches transcription of such erupted satellites not leaned against TADs and production of their DNA copies due to the action of TERT reverse transcriptase just like in telomeres and leads to insertion of telomere sequences into such SATs. Some sticky ends in satellites also add to chromosome instability.

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“…(3) Nucleases and polymerases participate in the processing of the DNA ends. 147 (4) The ligation of the processed DNA ends, performed by XLF, DNA ligase IV, and XRCC4 in NHEJ, 148 as shown in Figure 4 .…”

Section: Dna‐pkcs and Human Diseasesmentioning

confidence: 99%

The multifaceted functions of DNA‐PKcs: implications for the therapy of human diseases

Wu,

Song,

et al. 2024

MedComm

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The DNA‐dependent protein kinase (DNA‐PK), catalytic subunit, also known as DNA‐PKcs, is complexed with the heterodimer Ku70/Ku80 to form DNA‐PK holoenzyme, which is well recognized as initiator in the nonhomologous end joining (NHEJ) repair after double strand break (DSB). During NHEJ, DNA‐PKcs is essential for both DNA end processing and end joining. Besides its classical function in DSB repair, DNA‐PKcs also shows multifaceted functions in various biological activities such as class switch recombination (CSR) and variable (V) diversity (D) joining (J) recombination in B/T lymphocytes development, innate immunity through cGAS–STING pathway, transcription, alternative splicing, and so on, which are dependent on its function in NHEJ or not. Moreover, DNA‐PKcs deficiency has been proven to be related with human diseases such as neurological pathogenesis, cancer, immunological disorder, and so on through different mechanisms. Therefore, it is imperative to summarize the latest findings about DNA‐PKcs and diseases for better targeting DNA‐PKcs, which have shown efficacy in cancer treatment in preclinical models. Here, we discuss the multifaceted roles of DNA‐PKcs in human diseases, meanwhile, we discuss the progresses of DNA‐PKcs inhibitors and their potential in clinical trials. The most updated review about DNA‐PKcs will hopefully provide insights and ideas to understand DNA‐PKcs associated diseases.

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Stages, scaffolds and strings in the spatial organisation of non-homologous end joining: Insights from X-ray diffraction and Cryo-EM

Cited by 10 publications

References 135 publications

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Chromosome instability induced by mutations in TAD anchors leads to tumors

The multifaceted functions of DNA‐PKcs: implications for the therapy of human diseases

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