Non-homologous end joining (NHEJ), which is the predominant DNA double-strand break repair pathway in vertebrates, has been studied extensively, but the role of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is controversial. From genetic and cell-based experiments, DNA-PKcs is presumed to be a critical factor in DNA-end processing, and from structural experiments, DNA-PKcs facilitates DNA-end synapsis. However, these experiments are at odds with biochemical experiments that unequivocally show that DNA-PKcs inhibits NHEJ reactions. Thus, the precise function of DNA-PKcs is currently ambiguous. Here, we revisit this discrepancy using NHEJ reconstitution experiments. Under optimized conditions, we find that DNA-PKcs has a significant role in joining both cohesive and blunt ends in an ATP-dependent manner. Of note, the end joining efficiency of both cohesive vs. blunt ends is indistinguishable. These data suggest that DNA-PKcs promotes a unique productive synaptic complex that is driven in a manner independent of DNA-end structure.

Non-homologous end-joining (NHEJ) is a major double-stranded DNA (dsDNA) break repair pathway essential for V(D)J recombination during lymphocyte development. The Ku70/Ku80 heterodimer (Ku) initiates NHEJ by encircling dsDNA ends and recruiting DNA-PKcs. Ku70 in plants and mammals acquired a C-terminal SAP domain implicated in nucleic acid binding. Here, we show that in murine models, the SAP domain is dispensable for Ku stability and recruitment to DNA breaks. Unlike Ku70-/- mice, Ku70ΔSAP/ΔSAP mice exhibit normal lymphocyte development despite mild radiation sensitivity. Structural modeling places the SAP domain in adjacent DNA grooves, where it can restrict Ku's lateral movement along dsDNA. Correspondingly, in mice lacking DNA-PKcs that caps the ends, Ku70ΔSAP reduces T cell counts and deletion sizes, consistent with Ku translocating off DNA. Moreover, SAP deletion reduced DNA-end affinity, increased dissociation, and exchange of purified Ku at low concentrations, and increased multiple-loading at high concentrations, consistent with increased lateral movement. In DNA-PKcs-/- murine fibroblasts, deletion or lysine mutation (K593/4A, corresponding to K595/6A in human Ku70) in the SAP domain decreased the relative intensity of laser-induced Ku spots, revealing a role of the SAP domain in constraining Ku lateral movement on dsDNA in the absence of DNA-PKcs (or in the short-range complex).

Ovarian clear cell carcinoma (CCC) is a highly aggressive malignancy characterized by poor prognosis due to its resistance to chemotherapy, particularly in the advanced stages. This neoplasm originates from endometriotic cysts containing high levels of iron, derived from blood. This accumulation of iron generates oxidative stress, leading to DNA damage and carcinogenesis. Genomic analyses have identified frequent mutations in ARID1A and PIK3CA, which are also present in normal endometrial gland cells, suggesting that the cells originated from normal endometrium. A unique gene expression profile, termed the OCCC signature, is characterized by the upregulation of HNF1B signaling and oxidative stress-related genes. These genes are induced by the stressful microenvironment of endometriotic cysts. Comprehensive DNA methylation analysis has revealed that ovarian CCC exhibits a distinct epigenetic landscape, marked by global hypomethylation of HNF1B-related transcriptional signals, resulting in their activation. Functionally, ovarian CCC is characterized by enhanced anaerobic metabolism, mitochondrial dysfunction, and resistance to ferroptosis, an iron-dependent form of cell death. Notably, the suppression of HNF1B has been associated with increased sensitivity to chemotherapy, highlighting its role in drug resistance. These findings suggest that ovarian CCC develops through epigenomic carcinogenesis, wherein chronic oxidative stress in endometriotic cysts drives epigenetic alterations that promote tumor formation and therapeutic resistance, representing “epigenetic dispositions”. The elucidation of these mechanisms may provide novel insights into potential therapeutic targets to overcome the aggressive behaviors of ovarian CCC.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a “repair accident model” whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

Supplementary Data from Yin Yang 1 Modulates Taxane Response in Epithelial Ovarian Cancer

Functional annotations (from DAVID) for genes with higher expression in cells with low spheroid-forming capacity relative to cells with high spheroid-forming capacity.

Figures S1, S2 and Tables S1,S2 and S7

Human genomic DNA contains a number of diverse repetitive sequence motifs, often identified as fragile sites leading to genetic instability. Among them, expansion events occurring at triplet repeats have been extensively studied due to their association with neurological disorders, including Huntington’s disease (HD). In the case of HD, expanded CAG triplet repeats in the HTT gene are thought to cause the onset. The expansion of CAG triplet repeats is believed to be triggered by the emergence of stem-loops composed of CAG triplet repeats, while the underlying molecular mechanisms are largely unknown. Therefore, identifying proteins recruited on such stem loops would be useful to understand the molecular mechanisms leading to the genetic instability of CAG triplet repeats. We previously developed a plasmid DNA pull-down methodology that captures proteins specifically assembled on any sequence of interest using nuclear extracts. Analysis by Mass Spectrometry revealed that among the proteins specifically bound to a stem-loop composed of CAG triplet repeats, many turned out to belong to DNA repair pathways. We expect our data set to represent a useful entry point for the design of assays allowing the molecular mechanisms of genetic instability at CAG triplet repeats to be explored.

Differentially expressed genes that exhibit increased expression in the low spheroid-forming capacity cell lines that discriminate between high and low potential to survive serum-free spheroid culture.

Functional annotations (from the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8) for genes with higher expression in cells with high spheroid-forming capacity relative to cells with low spheroid-forming capacity.

Differentially expressed genes that exhibit increased expression in the high spheroid-forming capacity cell lines that discriminate between high and low potential to survive serum-free spheroid culture.

The cellular response to alkylation damage is complex, involving multiple DNA repair pathways and checkpoint proteins, depending on the DNA lesion, the cell type, and the cellular proliferation state. The repair of and response to O-alkylation damage, primarily O-6-methylguaine DNA adducts (O-6-mG), is the purview of O-6- methylguanine-DNA methyltransferase (MGMT). Alternatively, this lesion, if left un-repaired, induces replication-dependent formation of the O-6-mG:T mis-pair and recognition of this mis-pair by the post-replication mismatch DNA repair pathway (MMR). Two models have been suggested to account for MMR and O-6-mG DNA lesion dependent formation of DNA double-strand breaks (DSBs) and the resulting cytotoxicity - futile cycling and direct DNA damage signaling. While there have been hints at crosstalk between the MMR and base excision repair (BER) pathways, clear mechanistic evidence for such pathway coordination in the formation of DSBs has remained elusive. However, using a novel protein capture approach, Fuchs and colleagues have demonstrated that DSBs result from an encounter between MMR-induced gaps initiated at alkylation induced O-6-mG:C sites and BER-induced nicks at nearby N-alkylation adducts in the opposite strand. The accidental encounter between these two repair events is causal in the formation of DSBs and the resulting cellular response, documenting a third model to account for O-6-mG induced cell death in non-replicating cells. This graphical review highlights the details of this Repair Accident model, as compared to current models, and we discuss potential strategies to improve clinical use of alkylating agents such as temozolomide, that can be inferred from the Repair Accident model.

AbstractSpheroids exhibit drug resistance and slow proliferation, suggesting involvement in cancer recurrence. The protein kinase C inhibitor UCN-01 (7-hydroxystaurosporine) has shown higher efficacy against slow proliferating and/or quiescent ovarian cancer cells. In this study, tumorigenic potential was assessed using anchorage-independent growth assays and spheroid-forming capacity, which was determined with ovarian cancer cell lines as well as primary ovarian cancers. Of 12 cell lines with increased anchorage-independent growth, 8 formed spheroids under serum-free culture conditions. Spheroids showed reduced proliferation (P < 0.0001) and Ki-67 immunostaining (8% vs. 87%) relative to monolayer cells. Spheroid formation was associated with increased expression of mitochondrial pathway genes (P ≤ 0.001) from Affymetrix HT U133A gene expression data. UCN-01, a kinase inhibitor/mitochondrial uncoupler that has been shown to lead to Puma-induced mitochondrial apoptosis as well as ATP synthase inhibitor oligomycin, demonstrated effectiveness against spheroids, whereas spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal, whereas there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or oltipraz. Taken together, our findings suggest that genes involved in mitochondrial function in spheroids may be an important therapeutic target in preventing disease recurrence.

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O-6-methylguanine (O-6 mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O(6)mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O(6)mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMRdependent, repair synthesis. Our evidence also suggests that MMR, initiated at O(6)mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O-6 mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species.For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

In Japan, the standard procedure for radical hysterectomy is based on Okabayashi's approach reported in 1921.[1][1] A novel aspect of this technique is the discovery of the paravaginal space between the posterior (dorsal) leaf of the vesico-uterine ligament and vaginal blood vessels in the

The lesion bypass pathway, translesion synthesis (TLS), exists in essentially all organisms and is considered a pathway for postreplicative gap repair and, at the same time, for lesion tolerance. As with the saying “a trip is not over until you get back home,” studying TLS only at the site of the lesion is not enough to understand the whole process of TLS. Recently, a genetic study uncovered that polymerase V (Pol V), a poorly expressed Escherichia coli TLS polymerase, is not only involved in the TLS step per se but also participates in the gap-filling reaction over several hundred nucleotides.

The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.