Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

Uematsu, Naoya; Weterings, Eric; Yano, Keiji; Morotomi-Yano, Keiko; Jakob, Burkhard; Taucher-Scholz, G.; Mari, Pierre-Olivier; Gent, Dik C. van; Chen, Benjamin P.C.; Chen, David J.

doi:10.1083/jcb.200608077

Cited by 360 publications

(449 citation statements)

References 36 publications

(86 reference statements)

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“…2B). Likewise, DNA-PKcs autophosphorylation at Ser 2056 (27) increased when PEO1 cells were treated with ABT-888 (Fig S2A), and this phosphorylation was reversed by DNA-PK inhibition ( Fig. S2 B and C).…”

Section: Parp Inhibitor Synthetic Lethality Is Independent Of Xrcc1 Amentioning

confidence: 92%

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

Patel

Sarkaria

Kaufmann

2011

Proc. Natl. Acad. Sci. U.S.A.

478

431

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Poly(ADP-ribose) polymerase (PARP) inhibitors are strikingly toxic to cells with defects in homologous recombination (HR). The mechanistic basis for these findings is incompletely understood. Here, we show that PARP inhibitor treatment induces phosphorylation of DNA-dependent protein kinase substrates and stimulates error-prone nonhomologous end joining (NHEJ) selectively in HRdeficient cells. Notably, inhibiting DNA-dependent protein kinase activity reverses the genomic instability previously reported in these cells after PARP inhibition. Moreover, disabling NHEJ by using genetic or pharmacologic approaches rescues the lethality of PARP inhibition or down-regulation in cell lines lacking BRCA2, BRCA1, or ATM. Collectively, our results not only implicate PARP1 catalytic activity in the regulation of NHEJ in HR-deficient cells, but also indicate that deregulated NHEJ plays a major role in generating the genomic instability and cytotoxicity in HR-deficient cells treated with PARP inhibitors.chemotherapy | DNA repair | synthetic lethality | double-strand break repair P oly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that synthesizes poly(ADP-ribose) polymer when activated by DNA nicks or breaks. Activation of PARP1 has important effects on a variety of cellular processes, including base excision repair (BER), transcription, and cellular bioenergetics (1). The role of PARP1 in the DNA damage response sparked interest in the development of PARP inhibitors as potential chemosensitizers for the treatment of cancer (1, 2). The more recent observation that PARP inhibition is particularly lethal to cells deficient in homologous recombination (HR) proteins (3-8) has generated additional excitement in the cancer chemotherapy community. The current explanation for this hypersensitivity focuses on a mechanism (Fig. 1A) in which loss of PARP1 activity is thought to result in accumulation of DNA single-strand breaks (SSBs), which are subsequently converted to DNA double-strand breaks (DSBs) by the cellular replication and/or transcription machinery. These DSBs, which are repaired by HR in BRCApositive cells, are presumed to accumulate in BRCA1-or BRCA2-deficient cells, leading to subsequent cell death. Heightened sensitivity to PARP inhibition has also been observed in cells with other genetic lesions that affect HR, including phosphatase and tensin homolog (PTEN) deficiency (5), ataxia telangiectasia mutated (ATM) deficiency (7,8), and Aurora A overexpression (6).Although the preceding studies underscore the importance of PARP1 and HR in maintaining genomic stability, they do not address the role of nonhomologous end joining (NHEJ), an alternate DSB repair modality that directly joins broken ends of DNA with little or no regard for sequence homology (9). NHEJ is initiated when free DNA ends are bound by Ku70 and Ku80, which recruit the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The resulting complex, known as the DNAdependent protein kinase (DNA-PK) complex, phosphorylates downstream targe...

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Section: Parp Inhibitor Synthetic Lethality Is Independent Of Xrcc1 Amentioning

confidence: 92%

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

Patel

Sarkaria

Kaufmann

2011

Proc. Natl. Acad. Sci. U.S.A.

478

431

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“…Laser microirradiation was carried out with a pulsed nitrogen laser (Micropoint Ablation laser system; Photonic Instruments Inc.) coupled to a Zeiss Axiovert200M microscope and focused through a PlanApochromat 63ϫ/NA 1.40 oil immersion objective (44). The output laser power was set at 75% of maximum, which creates double-stranded breaks (DSBs) (as confirmed by ␥H2AX staining) and induces accumulation of EGFP-tagged RFWD3 in living cells.…”

Section: Methodsmentioning

confidence: 99%

RING Finger and WD Repeat Domain 3 (RFWD3) Associates with Replication Protein A (RPA) and Facilitates RPA-mediated DNA Damage Response

Liu

Chu

Yucer

et al. 2011

Journal of Biological Chemistry

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DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker ␥H2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.The cellular response to genotoxic stress initiates multiple signal transduction pathways that include transcription regulation, cell cycle arrest, DNA damage repair, and apoptosis (1, 2). Central to DDR 2 are two checkpoint kinases ATM and ATR that phosphorylate many downstream effectors to execute these functions (3, 4). Identification of key players and the characterization of their molecular functions enable a more thorough understanding of the DDR pathways, which are critical for maintaining genomic integrity. Several recent proteomic screens have drastically expanded the landscape of DDR pathways with the identification of hundreds of putative ATM/ATR substrates (5-7). Recently, we investigated the role of a previously uncharacterized protein, RING finger and WD repeat domain 3 (RFWD3, also known as RNF201 and FLJ10520; GenBank TM number 55159) in DDR (5, 8). Originally identified from a proteomic screen for ATM/ATR substrates, RFWD3 was found phosphorylated at several conserved SQ sites in cells that were treated with ionizing radiation (IR) and replication blocking agents. Subsequent biochemical analysis revealed that RFWD3 can form a complex with MDM2 and p53 and is required for the maintenance of high levels of p53 upon DNA damage induction. The RFWD3 protein contains several well characterized functional domains as well as domains of unknown functions. The N-terminal RING finger domain is a conserved E3 ubiquitin (Ub) ligase domain. In vitro r...

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“…Live cell imaging of DNA repair proteins induced with a micro-laser is now widely used to study the kinetics of DNA damage responses [38][39][40] . Live cell imaging with heavy ions has shown that DNA repair proteins are recruited at sites of heavy ion hits in cell nuclei of fibroblast cells within seconds, and no large scale chromatin movements are associated to the repair activity 41 , yet some movement is observed in the repair protein foci.…”

Section: Dna Damagementioning

confidence: 99%

Heavy ion carcinogenesis and human space exploration

Durante¹,

Cucinotta²

2008

Nat Rev Cancer

502

324

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Prior to the human exploration of Mars or long duration stays on the Earth's moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets. Introduction:

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Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

Cited by 360 publications

References 36 publications

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

RING Finger and WD Repeat Domain 3 (RFWD3) Associates with Replication Protein A (RPA) and Facilitates RPA-mediated DNA Damage Response

Heavy ion carcinogenesis and human space exploration

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