Browsing by Subject "DNA Breaks, Double-Stranded"

Now showing 1 - 5 of 5

Open Access
A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest.
(Cell reports, 2020-08) Tsabar, Michael; Mock, Caroline S; Venkatachalam, Veena; Reyes, Jose; Karhohs, Kyle W; Oliver, Trudy G; Regev, Aviv; Jambhekar, Ashwini; Lahav, Galit
Cellular responses to stimuli can evolve over time, resulting in distinct early and late phases in response to a single signal. DNA damage induces a complex response that is largely orchestrated by the transcription factor p53, whose dynamics influence whether a damaged cell will arrest and repair the damage or will initiate cell death. How p53 responses and cellular outcomes evolve in the presence of continuous DNA damage remains unknown. Here, we have found that a subset of cells switches from oscillating to sustained p53 dynamics several days after undergoing damage. The switch results from cell cycle progression in the presence of damaged DNA, which activates the caspase-2-PIDDosome, a complex that stabilizes p53 by inactivating its negative regulator MDM2. This work defines a molecular pathway that is activated if the canonical checkpoints fail to halt mitosis in the presence of damaged DNA.
Open Access
DEK is required for homologous recombination repair of DNA breaks.
(Scientific reports, 2017-03-20) Smith, Eric A; Gole, Boris; Willis, Nicholas A; Soria, Rebeca; Starnes, Linda M; Krumpelbeck, Eric F; Jegga, Anil G; Ali, Abdullah M; Guo, Haihong; Meetei, Amom R; Andreassen, Paul R; Kappes, Ferdinand; Vinnedge, Lisa M Privette; Daniel, Jeremy A; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I
DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.
Open Access
Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.
(G3 (Bethesda, Md.), 2020-10) Gamble, Dionna; Shaltz, Samantha; Jinks-Robertson, Sue
Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' vs. 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' vs. 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.
Open Access
Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors.
(Cell Cycle, 2013-03-15) Weingeist, David M; Ge, Jing; Wood, David K; Mutamba, James T; Huang, Qiuying; Rowland, Elizabeth A; Yaffe, Michael B; Floyd, Scott; Engelward, Bevin P
A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.
Open Access
Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.
(Proc Natl Acad Sci U S A, 2013-12-17) Chen, Chen; Zhang, Liguo; Huang, Nai-Jia; Huang, Bofu; Kornbluth, Sally
Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.