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DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao et al. ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)–free, short-range state to align the DSB ends (Chen et al. ). Using single-particle cryo–electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.

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Supplement Figure S4. Expression of Nrf2 and ERα in MCF-7Ca, LTLTCa and LTLTCa+Nrf2shRNA (LTLTCa-Nrf2KD) cells.

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Suppl. Table 1: IC50 values for the DNA Ligase inhibitor L67 for three cancer cells lines, a telomerase-immortalized fibroblast and the Rho minus derivatives of each of the cell lines; Suppl. Table 2: DNA sequences of primers used for amplification of specific fragments and nuclear and mitochondrial DNA using the polymerase chain reaction; Suppl. Table 3: Reaction conditions used to specific fragments and nuclear and mitochondrial DNA by the polymerase chain reaction

Panel A. Effect of L67 on the cell cycle distribution of synchronized HCA-Ltrt cells. Panel B. Effect of L67 on mitochondrial superoxide level in HCA-Ltrt cells and HCA-Ltrt Rho minus cells

Panel A. Induction of gammaH2AX foci in HeLa cells, HeLa Rho minus cells and HeLa cells expressing mitochondrially targeted E. coli DNA ligase by L67. Panel B. N-acetyl cysteine inhibits L67-induced autophosphorylation of ATM in HeLa cells.

Supplementary Materials and methods plus legends for six supplementary figures

Panel A. Characterization of HeLA cells expressing mitochondrially-targeted E. coli DNA ligase. DNA encoding the E. coli DNA detected by PCR and expression of GFP-tagged E. coli DNA ligase detected by immunoblotting. Panel B. Effect of L67 on DNA ligation by purified DN Ligase IIIalpha/XRCC1 and E.coli DNA ligase

L67 does not induce cleavage of caspases 7 or 9 in HeLa cells.

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1–dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431–41. ©2016 AACR.

Supplementary Figure 1 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]

Supplementary Figure 5 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 2 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 4 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Methods and Materials from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

Supplementary Figure 3 from Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair

DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.