Editor's Note: Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway.

<div>Abstract<p>Werner syndrome is genetically linked to mutations in <i>WRN</i> that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2<sup>−/−</sup> cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia–telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2<sup>−/−</sup> cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia–deficient tumors by their sensitization to DNA cross-linking agents is discussed. <i>Cancer Res; 73(17); 5497–507. ©2013 AACR</i>.</p></div>

<div>Abstract<p>Werner syndrome is genetically linked to mutations in <i>WRN</i> that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2<sup>−/−</sup> cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia–telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2<sup>−/−</sup> cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia–deficient tumors by their sensitization to DNA cross-linking agents is discussed. <i>Cancer Res; 73(17); 5497–507. ©2013 AACR</i>.</p></div>

Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2(-/-) cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia-telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2(-/-) cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia-deficient tumors by their sensitization to DNA cross-linking agents is discussed.

<p>PDF file, 1010K, Chemical structures of structurally related analogs of the previously identified parent compound NSC 19630 (S1); Effect of NSC 617145 on WRN catalytic functions and DNA unwinding by other helicases (S2); Effect of selected small molecules structurally related to NSC 19630 on HeLa cell proliferation (S3); NSC 617145 inhibits cell proliferation in a WRN-dependent manner (S4); Effect of NSC 617145 on WRN-depleted cells expressing ectopic WRN protein that is active or inactive as for ATPase/helicase activity (S5); Specificity of WRN helicase inhibition by NSC 617145 (S6); NSC 617145 induces DNA damage (S7); NSC 617145 induces apoptosis when WRN is present (S8); Elevated PCNA staining of NSC 617145-treated cells is WRNdependent (S9); Effect of NSC 617145 exposure on cell cycle (S10); NSC 617145 exposure does not sensitize HeLa, FA-A, or FA-D2 cells to hydroxyurea (S11); WRN depletion does not affect MMC sensitivity of FA-D2 mutant cells (S12); Western blot analysis of ATM activation in FA-D2 mutant cells (S13).</p>

<p>PDF file, 1010K, Chemical structures of structurally related analogs of the previously identified parent compound NSC 19630 (S1); Effect of NSC 617145 on WRN catalytic functions and DNA unwinding by other helicases (S2); Effect of selected small molecules structurally related to NSC 19630 on HeLa cell proliferation (S3); NSC 617145 inhibits cell proliferation in a WRN-dependent manner (S4); Effect of NSC 617145 on WRN-depleted cells expressing ectopic WRN protein that is active or inactive as for ATPase/helicase activity (S5); Specificity of WRN helicase inhibition by NSC 617145 (S6); NSC 617145 induces DNA damage (S7); NSC 617145 induces apoptosis when WRN is present (S8); Elevated PCNA staining of NSC 617145-treated cells is WRNdependent (S9); Effect of NSC 617145 exposure on cell cycle (S10); NSC 617145 exposure does not sensitize HeLa, FA-A, or FA-D2 cells to hydroxyurea (S11); WRN depletion does not affect MMC sensitivity of FA-D2 mutant cells (S12); Western blot analysis of ATM activation in FA-D2 mutant cells (S13).</p>

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

<p>PDF file, 108K.</p>

<p>PDF file, 108K.</p>

<p>PDF file, 106K, Supplemental information regarding proteins, DNA substrates, immunofluorescence, cell cycle analysis by flow cytometry, and additional experimental procedures.</p>

<p>PDF file, 106K, Supplemental information regarding proteins, DNA substrates, immunofluorescence, cell cycle analysis by flow cytometry, and additional experimental procedures.</p>

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.

Histone ubiquitylation and its roles in transcription and DNA damage response.

Involvement of serine / threonine protein kinases in DNA damage response and cell division in bacteria