Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

A Cell System-Assisted Strategy for Evaluating the Natural Antioxidant-Induced Double-Stranded DNA Break (DSB) Style.

Natural antioxidants derived from plants exert various physiological effects, including antitumor effects. However, the molecular mechanisms of each natural antioxidant have not yet been fully elucidated. Identifying the targets of natural antioxidants with antitumor properties in vitro is costly and time-consuming, and the results thus obtained may not reliably reflect in vivo conditions. Therefore, to enhance understanding regarding the antitumor effects of natural antioxidants, we focused on DNA, one of the targets of anticancer drugs, and evaluated whether antioxidants, e.g., sulforaphane, resveratrol, quercetin, kaempferol, and genistein, which exert antitumor effects, induce DNA damage using gene-knockout cell lines derived from human Nalm-6 and HeLa cells pretreated with the DNA-dependent protein kinase inhibitor NU7026. Our results suggested that sulforaphane induces single-strand breaks or DNA strand crosslinks and that quercetin induces double-strand breaks. In contrast, resveratrol showed the ability to exert cytotoxic effects other than DNA damage. Our results also suggested that kaempferol and genistein induce DNA damage via unknown mechanisms. Taken together, the use of this evaluation system facilitates the analysis of the cytotoxic mechanisms of natural antioxidants.

Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

Complex genetic interactions between DNA polymerase ß and the NHEJ ligase.

Mammalian cells possess multiple pathways for repairing various types of DNA damage. Although the molecular mechanisms of each DNA repair pathway have been analyzed by biochemical analysis and cell biological analysis, interplay between different pathways has not been fully elucidated. In this study, using human Nalm-6-mutant cell lines, we analyzed the relationship between the base excision repair factor DNA polymerase ß (POLß) and DNA ligase IV (LIG4), which is essential for DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). We found that cells lacking both POLß and LIG4 grew significantly more slowly than either single mutant, indicating cooperative functions of the two proteins in normal cell growth. To further investigate the genetic interaction between POLß and LIG4, we examined DNA damage sensitivity of the mutant cell lines. Our results suggested that NHEJ acts as a backup pathway for repairing alkylation damage (when converted into DSBs) in the absence of POLß. Surprisingly, despite the critical role of POLß in alkylation damage repair, cells lacking POLß exhibited increased resistance to camptothecin (a topoisomerase I inhibitor that induces DNA single-strand breaks), irrespective of the presence or absence of LIG4. A LIG4-independent increased resistance associated with POLß loss was also observed with ionizing radiation; however, cells lacking both POLß and LIG4 were more radiosensitive than either single mutant. Taken together, our findings provide novel insight into the complex interplay between different DNA repair pathways.

GPR31 and GPR151 are activated under acidic conditions.

Recent studies have revealed that not only proton-sensing channels, but also one family of G protein-coupled receptors (GPCRs) comprising OGR1, GPR4, G2A and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31 and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31-Giα and GPR151-Giα fusion proteins in a narrow range between pH 6 and 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at pH ∼5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

A novel partial agonist of GPBA reduces blood glucose level in a murine glucose tolerance test.

GPBA is a G protein-coupled receptor that is activated by bile acids. Because activation of GPBA leads to increased cAMP levels and secretion of incretins and insulin, GPBA has been proposed as a promising drug target for the treatment of metabolic syndrome. Previously, we have developed a ligand-screening system to identify novel agonists of GPBA by means of a fusion protein of GPBA with G protein stimulatory α subunit (Gsα) and by a [35S]GTPγS-binding assay. To express the GPBA-Gsα fusion protein, transgenic silkworms were employed in this study, and cell membrane fractions were prepared from their fat body or pupae. We applied them to the screening of a chemical library that contains 10,625 compounds from the RIKEN Natural Products Depository (NPDepo). Eventually, a unique partial agonist, GUM2, was successfully identified. Our results indicated that the GPCR-Gα fusion proteins were beneficial for ligand identification and that the transgenic silkworms were useful for large-scale production of GPCRs. In HEK293 cells transiently expressing GPBA, GUM2 showed 50% effective concentration (EC50) of 3.5 ± 2.4µM and induced GPBA internalization as effectively as did an endogenous agonist, TLC. We also confirmed that GUM2 stimulates insulin secretion in MIN6 cells. Moreover, a single 2mg/kg dose of GUM2 significantly reduced blood glucose levels in mice during an intraperitoneal glucose tolerance test even though GUM2 is only a partial agonist with a low intrinsic activity. We concluded that GUM2 is a good candidate for research on GPBA signaling under physiological conditions and for the development of GPBA-targeting therapeutic compounds.

Mechanistic basis for increased human gene targeting by promoterless vectors-roles of homology arms and Rad54 paralogs.

Gene targeting by homologous recombination provides the definitive tool for analyzing gene function. Promoterless vectors, which do not possess a promoter to drive marker gene expression, confer higher targeting efficiencies than conventional vectors due to the reduced number of drug-resistant clones. We here show that gene-targeting efficiency is typically ≥ 25% with the use of exon-trapping-type promoterless vectors in a human diploid cell line, Nalm-6. The efficiency of exon-trapping gene targeting was correlated with the level of target gene expression when a 2A peptide sequence was linked to the marker gene. Intriguingly, total arm length was not necessarily a determinant of targeting efficiency, as longer arms tend to enhance both homologous (targeted) and nonhomologous (nontargeted) integration of the vector; rather, the presence of an exon in the 5' arm led to a decreased targeting efficiency. Strikingly, loss of Rad54 did not severely affect the targeting efficiency of exon-trap vectors. Moreover, additional deletion of the Rad54 paralog Rad54B had limited impact on the high-efficiency gene targeting. These results indicate that targeted integration occurs in human cells even when both Rad54 and Rad54B are missing. These studies provide additional important insight into the contribution of various DNA repair factors on the targeting mechanics.

Mutations in XRCC4 cause primordial dwarfism without causing immunodeficiency.

In successive reports from 2014 to 2015, X-ray repair cross-complementing protein 4 (XRCC4) has been identified as a novel causative gene of primordial dwarfism. XRCC4 is indispensable for non-homologous end joining (NHEJ), the major pathway for repairing DNA double-strand breaks. As NHEJ is essential for V(D)J recombination during lymphocyte development, it is generally believed that abnormalities in XRCC4 cause severe combined immunodeficiency. Contrary to expectations, however, no overt immunodeficiency has been observed in patients with primordial dwarfism harboring XRCC4 mutations. Here, we describe the various XRCC4 mutations that lead to disease and discuss their impact on NHEJ and V(D)J recombination.

Identification and molecular docking studies for novel inverse agonists of SREB, super conserved receptor expressed in brain.

The identification of novel synthetic ligands for G protein-coupled receptors (GPCRs) is important not only for understanding human physiology, but also for the development of novel drugs, especially for orphan GPCRs for which endogenous ligands are unknown. One of the orphan GPCR subfamilies, Super conserved Receptor Expressed in Brain (SREB), consists of GPR27, GPR85 and GPR173 and is expressed in the central nervous system. We report herein the identification of inverse agonists for the SREB family without their agonists. We carried out an in vitro screening of 5472 chemical compounds from the RIKEN NPDepo chemical library. The binding of [(35) S]GTPγS to the GPR173-Gsα fusion protein expressed in Sf9 cells was measured and resulted in the identification of 8 novel GPR173 inverse agonists. The most potent compound showed an IC50 of approximately 8 µm. The identified compounds were also antagonists for other SREB members, GPR27 and GPR85. These results indicated that the SREB family could couple Gs-type G proteins, and SREB-Gsα fusion proteins showed significant constitutive activities. Moreover, a molecular model of GPR173 was constructed using the screening results. The combination of computational and biological methods will provide a unique approach to ligand identification for orphan GPCRs and brain research.

In vivo and in vitro evaluation of novel µ-opioid receptor agonist compounds.

Opioids are the most effective and widely used drugs for pain treatment. Morphine is an archetypal opioid and is an opioid receptor agonist. Unfortunately, the clinical usefulness of morphine is limited by adverse effects such as analgesic tolerance and addiction. Therefore, it is important to study the development of novel opioid agonists as part of pain control. The analgesic effects of opioids are mediated by three opioid receptors, namely opioid µ-, δ-, and κ-receptors. They belong to the G protein-coupled receptor superfamily and are coupled to Gi proteins. In the present study, we developed a ligand screening system to identify novel opioid µ-receptor agonists that measures [(35)S]GTPγS binding to cell membrane fractions prepared from the fat body of transgenic silkworms expressing µ-receptor-Gi1α fusion protein. We screened the RIKEN Natural Products Depository (NPDepo) chemical library, which contains 5848 compounds, and analogs of hit compounds. We successfully identified a novel, structurally unique compound, that we named GUM1, with agonist activity for the opioid µ-receptor (EC50 of 1.2 µM). The Plantar Test (Hargreaves' Method) demonstrated that subcutaneous injection of 3mg/kg of GUM1 into wild-type rats significantly extended latency time. This extension was also observed in a rat model of morphine tolerance and was inhibited by pre-treatment of naloxone. The unique molecular skeleton of GUM1 makes it an attractive molecule for further ligand-opioid receptor binding studies.

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.

Analysis of the role of homology arms in gene-targeting vectors in human cells.

Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

Interference in DNA replication can cause mitotic chromosomal breakage unassociated with double-strand breaks.

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/)KU70(-/-) DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/)LIG4(-/-) Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.

Both CpG methylation and activation-induced deaminase are required for the fragility of the human bcl-2 major breakpoint region: implications for the timing of the breaks in the t(14;18) translocation.

The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.

Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs.

Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis-Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA-protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.

Nucleofection-based gene targeting in human pre-B cells.

Electroporation is a powerful and convenient means for transfection of nonviral vectors into mammalian cells, providing an essential tool for numerous applications including gene targeting via homologous recombination. Recent evidence clearly suggests that high-efficiency gene transfer can be achieved in most cell lines by nucleofection, an electroporation-based transfection method that allows transfected vectors to directly enter the nucleus. In this paper, we analyze the effectiveness of nucleofection for gene targeting using human pre-B cells. For this, we tested 93 different transfection conditions, and found several conditions that gave high (~80%) transfection efficiency with low cytotoxicity (~70% survival rate). Remarkably, under the optimal nucleofection conditions, the gene-targeting efficiency was ~2-5-fold higher than that achieved with conventional electroporation methods. We also found that nucleofection conditions with high transfection efficiency and low cytotoxicity tend to provide high gene-targeting efficiency. Our results provide significant implications for gene targeting, and suggest that nucleofection-based nonviral gene transfer is useful for systematic generation of human gene-knockout cell lines.

Functions and regulation of Artemis: a goddess in the maintenance of genome integrity.

Artemis is a structure-specific endonuclease when associated with and phosphorylated by DNA-dependent protein kinase catalytic subunit. This structure-specific endonuclease is responsible for the resolution of hairpin coding ends in V(D)J recombination. In DNA double-strand break repair, Artemis is implicated in the end-processing step of the non-homologous end-joining (NHEJ) pathway. Recently, we have demonstrated that the involvement of Artemis in NHEJ depends on the type of DNA damage. Interestingly, recent evidence suggests that the end-processing activity is not the only function of Artemis. Indeed, Artemis is rapidly phosphorylated by ataxia telangiectasia mutated in response to DNA damage, and such phosphorylation of Artemis appears to be involved in the regulation of cell cycle checkpoints. These findings suggest that Artemis is a multifunctional protein participating in the maintenance of genome integrity at two distinct levels; one at the end processing step of NHEJ, and the other at the signaling pathway of cell cycle regulation. Therefore, understanding Artemis function may give us profound insights into the DNA repair network. In this review, we summarize the functions and regulation of Artemis.

Heterozygous disruption of the DNA topoisomerase I gene confers cellular resistance to camptothecin in human cells.

DNA topoisomerase I (Top1) is a ubiquitous nuclear enzyme that plays essential roles in various cellular processes, such as transcription or replication. Agents that target Top1, involving camptothecin and its derivatives, are among the most effective anticancer drugs used in the clinic. Previous work has suggested that the level of Top1 expression correlates with the cytotoxicity of camptothecin, but no direct evidence has been provided thus far in the context of human cells with a strictly isogenic genetic background. In this study, we perform heterozygous disruption of the Top1 gene (TOP1) by gene targeting in a human pre-B cell line, Nalm-6, which is karyotypically stable and normal for p53 status. We show that the heterozygous loss of the TOP1 gene does confer cellular resistance to camptothecin, to an extent comparable to that observed in the absence of functional p53 protein. Such a tolerance was not observed with other agents that target DNA topoisomerase II. Our results provide direct evidence that human cells with decreased Top1 levels are significantly more resistant to killing by camptothecin than are otherwise isogenic cells.

Topoisomerase IIalpha inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line.

DNA transfection can be too inefficient to establish a desired number of stable transfectants, particularly in lymphocytes; however, this could be circumvented by increasing the absolute frequency of random integration. In this paper, we show that treating cells with topoisomerase II inhibitor following electroporation greatly (approximately 10- to 20-fold) enhances random integration of input DNA in a human pre-B lymphocyte cell line, Nalm-6. With the use of various kinds of topoisomerase II-targeting agents, we also present evidence that topoisomerase IIalpha inhibition is critical for the enhancement of random integration, while the contribution of topoisomerase IIbeta may be negligible. As topoisomerase IIalpha is highly expressed in vigorously growing cells, our results show that topoisomerase IIalpha inhibition provides a promising way of enhancing random integration in virtually all cultured cell lines.