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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 double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 A apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules. Double-strand DNA break repair by the non-homologous end joining pathway involves the transition from a complex that bridges the DNA ends to a complex that aligns the DNA for ligation through the dissociation of the kinase subunits of the DNA-PK complexes.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5′-phosphoryl and 3′-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown. Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells. Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids. Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold., Graphical Abstract Graphical AbstractIntegrative modeling from EM and SAXS with atomic resolution domain structures reveals the functional compaction of the XRCC1-DNA ligase IIIα complex that extends beyond the interacting C-terminal BRCT–BRCT domains to the catalytic region of DNA ligase IIIα.

To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in 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.

Homologous recombination/end joining (HR/HEJ)-deficient cancers with BRCA mutations utilize alternative DNA double-strand break repair pathways, particularly alternative non-homologous end joining or microhomology-mediated end joining (alt-EJ/MMEJ) during S and G2 cell cycle phases. Depletion of alt-EJ factors, including XRCC1, PARP1 and POLQ, is synthetically lethal with BRCA2 deficiency; yet, XRCC1 roles in HR-deficient cancers and replication stress are enigmatic. Here, we show that after replication stress, XRCC1 forms an active repair complex with POLQ and MRE11 that supports alt-EJ activity in vitro. BRCA2 limits XRCC1 recruitment and repair complex formation to suppress alt-EJ at stalled forks. Without BRCA2 fork protection, XRCC1 enables cells to complete DNA replication at the expense of increased genome instability by promoting MRE11-dependent fork resection and restart. High XRCC1 and MRE11 gene expression negatively impacts Kaplan–Meier survival curves and hazard ratios for HR-deficient breast cancer patients in The Cancer Genome Atlas. The additive effects of depleting both BRCA2 and XRCC1 indicate distinct pathways for replication restart. Our collective data show that XRCC1-mediated processing contributes to replication fork degradation, replication restart and chromosome aberrations in BRCA2-deficient cells, uncovering new roles of XRCC1 and microhomology-mediated repair mechanisms in HR-deficient cancers, with implications for chemotherapeutic strategies targeting POLQ and PARP activities.

Alternative end-joining (a-EJ) pathways, which repair DNA double-strand breaks (DSBs), are initiated by end resection that generates 3' single strands. This reaction is shared, at least in part, with homologous recombination but distinguishes a-EJ from the major nonhomologous end-joining pathway. Although the a-EJ pathways make only a minor and poorly understood contribution to DSB repair in nonmalignant cells, there is growing interest in these pathways, as they generate genomic rearrangements that are hallmarks of cancer cells. Here, we review and discuss the current understanding of the mechanisms and regulation of a-EJ pathways, the role of a-EJ in human disease, and the potential utility of a-EJ as a therapeutic target in cancer.

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.

DNA double-strand breaks (DSBs) are repaired by either the non-homologous end joining (NHEJ) or homologous recombination (HR) pathway. Pathway choice is determined by the generation of 3΄ single-strand DNA overhangs at the break that are initiated by the action of the Mre11–Rad50–Xrs2 (MRX) complex to direct repair toward HR. DSB repair occurs in the context of chromatin, and multiple chromatin regulators have been shown to play important roles in the repair process. We have investigated the role of the SWI/SNF ATP-dependent nucleosome-remodeling complex in the repair of a defined DNA DSB. SWI/SNF was previously shown to regulate presynaptic events in HR, but its function in these events is unknown. We find that in the absence of functional SWI/SNF, the initiation of DNA end resection is significantly delayed. The delay in resection initiation is accompanied by impaired recruitment of MRX to the DSB, and other functions of MRX in HR including the recruitment of long-range resection factors and activation of the DNA damage response are also diminished. These phenotypes are correlated with a delay in the eviction of nucleosomes surrounding the DSB. We propose that SWI/SNF orchestrates the recruitment of a pool of MRX that is specifically dedicated to HR.

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin.

© 2016, American Society for Biochemistry and Molecular Biology Inc. All rights reserved. DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.

Besides critical and well‐studied roles in maintaining genome stability, DNA repair pathways including base excision repair ( BER ) are also employed by mammalian cells for targeted events such as the generation of diverse antibody repertoires and the differentiation of pluripotent cells. In this issue of The EMBO Journal, Steinacher et al (2019) provide novel mechanistic insights into the complex role of SUMO ylation of key BER proteins during active DNA demethylation—a role they demonstrate to be important for cellular differentiation and that has broader implications for BER as a whole.

More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5’ end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.

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.

In contrast to a published report, reaction of 4,5-diamino-6-hydroxy-2-mercaptopyrimidine with 2 equiv of aromatic aldehydes produces a mixture of 6,7-diaryl-2-thioxopteridine-4-one and 6,7-diaryl-2-thioxo-7,8-dihydropteridine-4-one rather than a diimine. These compounds represent the correct structure for SCR7, a substance reported to be an inhibitor of nonhomologous end-joining, a DNA repair pathway. The dihydropteridine can be isolated as a minor product, and it can be oxidized to the pteridine.

DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.

Typically disease-causing CAG/CTG repeats expand, but rare affected families can display high levels of contraction of the expanded repeat amongst offspring. Understanding instability is important since arresting expansions or enhancing contractions could be clinically beneficial. The MutSβ mismatch repair complex is required for CAG/CTG expansions in mice and patients. Oddly, by unknown mechanisms MutSβ-deficient mice incur contractions instead of expansions. Replication using CTG or CAG as the lagging strand template is known to cause contractions or expansions respectively; however, the interplay between replication and repair leading to this instability remains unclear. Towards understanding how repeat contractions may arise, we performed in vitro SV40-mediated replication of repeat-containing plasmids in the presence or absence of mismatch repair. Specifically, we separated repair from replication: Replication mediated by MutSβ- and MutSα-deficient human cells or cell extracts produced slipped-DNA heteroduplexes in the contraction- but not expansion-biased replication direction. Replication in the presence of MutSβ disfavoured the retention of replication products harbouring slipped-DNA heteroduplexes. Post-replication repair of slipped-DNAs by MutSβ-proficient extracts eliminated slipped-DNAs. Thus, a MutSβ-deficiency likely enhances repeat contractions because MutSβ protects against contractions by repairing template strand slip-outs. Replication deficient in LigaseI or PCNA-interaction mutant LigaseI revealed slipped-DNA formation at lagging strands. Our results reveal that distinct mechanisms lead to expansions or contractions and support inhibition of MutSβ as a therapeutic strategy to enhance the contraction of expanded repeats.

Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression. ispartof: NATURE COMMUNICATIONS vol:9 issue:1 ispartof: location:England status: published

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

5-Fluorouracil (FU) is a halogenated nucleobase analog that is widely used in chemotherapy. Here we show that 5-hydroxymethyl-2′-deoxyuridine (hmUdR) synergistically enhances the activity of FU in cell lines derived from solid tumors but not normal tissues. While the cytotoxicity of FU and hmUdR was not directly related to the amount of the modified bases incorporated into cellular DNA, incubation with this combination resulted in dramatic increase in the number of single strand breaks in replicating cancer cells, leading to NAD-depletion as consequence of poly(ADP-ribose) synthesis and S phase arrest. Cell death resulting from the base/nucleoside combination did not occur by apoptosis, autophagy or necroptosis. Instead, the cells die via necrosis as a result of NAD depletion. The FU-related nucleoside analog, 5-fluoro-2′-deoxyuridine, also displayed synergy with hmUdR, whereas hmUdR could not be replaced by 5-hydroxymethyluracil. Among other 5-modified deoxyuridine analogs tested, 5-formyl-2′-deoxyuridine and, to a lesser extent, 5-hydroxy-2′-deoxyuridine, also acted synergistically with FU, whereas 5-hydroxyethyl-2′-deoxyuridine did not. Together, our results have revealed an unexpected synergistic interaction between deoxyuridine analogs and FU in a cancer cell-specific manner, and suggest that these novel base/nucleoside combinations could be developed into improved FU-based chemotherapies.

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines.

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines.

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.