Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Drug resistance mechanisms and treatment strategies mediated by Ubiquitin-Specific Proteases (USPs) in cancers: new directions and therapeutic options.

Drug resistance represents a significant obstacle in cancer treatment, underscoring the need for the discovery of novel therapeutic targets. Ubiquitin-specific proteases (USPs), a subclass of deubiquitinating enzymes, play a pivotal role in protein deubiquitination. As scientific research advances, USPs have been recognized as key regulators of drug resistance across a spectrum of treatment modalities, including chemotherapy, targeted therapy, immunotherapy, and radiotherapy. This comprehensive review examines the complex relationship between USPs and drug resistance mechanisms, focusing on specific treatment strategies and highlighting the influence of USPs on DNA damage repair, apoptosis, characteristics of cancer stem cells, immune evasion, and other crucial biological functions. Additionally, the review highlights the potential clinical significance of USP inhibitors as a means to counter drug resistance in cancer treatment. By inhibiting particular USP, cancer cells can become more susceptible to a variety of anti-cancer drugs. The integration of USP inhibitors with current anti-cancer therapies offers a promising strategy to circumvent drug resistance. Therefore, this review emphasizes the importance of USPs as viable therapeutic targets and offers insight into fruitful directions for future research and drug development. Targeting USPs presents an effective method to combat drug resistance across various cancer types, leading to enhanced treatment strategies and better patient outcomes.

Glycyrrhizic acid inhibits DNA damage repair and enhances cisplatin-induced apoptosis of melanoma cells.

This research was designed to prospect the mechanism and impact of glycyrrhizic acid (GA) on DNA damage repair and cisplatin (CP)-induced apoptosis of melanoma cells. First, human melanoma cell SK-MEL-28 was stimulated using GA for 24, 48, and 72 h. Then, the optimal treatment time and dosage were selected. After that, cell counting kit-8 (CCK-8) was employed for testing the cell viability, flow cytometry for the apoptosis, comet assay for the DNA damage of cells, and western blot for the cleaved-Caspase3, Caspase3, Bcl-2, and γH2AX protein expression levels. The experimental outcomes exhibited that as the GA concentration climbed up, the SK-MEL-28 cell viability dropped largely, while the apoptosis level raised significantly, especially at the concentration of 100 µm. In addition, compared with GA or CPtreatment only, CP combined with GA notably suppressed the viability of melanoma cells and promoted cell apoptosis at the cytological level. At the protein level, the combined treatment notably downregulated the Bcl-2 and Caspase3 expression levels, while significantly upregulated the cleaved-Caspase3 and γH2AX expression levels. Besides, CP + GA treatment promoted DNA damage at the DNA molecular level. Collectively, both GA and CP can inhibit DNA damage repair and enhance the apoptosis of SK-MEL-28 cells, and the synergistic treatment of both exhibits better efficacy.

Iron (II)-based metal-organic framework nanozyme for boosting tumor ferroptosis through inhibiting DNA damage repair and system Xc.

Development of ferroptosis-inducible nanoplatforms with high efficiency and specificity is highly needed and challenging in tumor ferrotherapy. Here, we demonstrate highly effective tumor ferrotherapy using iron (II)-based metal-organic framework (FessMOF) nanoparticles, assembled from disulfide bonds and ferrous ions. The as-prepared FessMOF nanoparticles exhibit peroxidase-like activity and pH/glutathione-dependent degradability, which enables tumor-responsive catalytic therapy and glutathione depletion by the thiol/disulfide exchange to suppress glutathione peroxidase 4, respectively. Upon PEGylation and Actinomycin D (ActD) loading, the resulting FessMOF/ActD-PEG nanoplatform induces marked DNA damage and lipid peroxidation. Concurrently, we found that ActD can inhibit Xc- system and elicit ferritinophagy, which further boosts the ferrotherapeutic efficacy of the FessMOF/ActD-PEG. In vivo experiments demonstrate that our fabricated nanoplatform presents excellent biocompatibility and a high tumor inhibition rate of 91.89%.

Metagenome mining and functional analysis reveal oxidized guanine DNA repair at the Lost City Hydrothermal Field.

The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H2 and low levels of O2 in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome-assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O2 levels rose following the great oxidation event.

Radiation-induced DNA double-strand breaks in cortisol exposed fibroblasts as quantified with the novel foci-integrated damage complexity score (FIDCS).

Without the protective shielding of Earth's atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.

LSD2 Is an Epigenetic Player in Multiple Types of Cancer and Beyond.

Histone demethylases, enzymes responsible for removing methyl groups from histone proteins, have emerged as critical players in regulating gene expression and chromatin dynamics, thereby influencing various cellular processes. LSD2 and LSD1 have attracted considerable interest among these demethylases because of their associations with cancer. However, while LSD1 has received significant attention, LSD2 has not been recognized to the same extent. In this study, we conduct a comprehensive comparison between LSD2 and LSD1, with a focus on exploring LSD2's implications. While both share structural similarities, LSD2 possesses unique features as well. Functionally, LSD2 shows diverse roles, particularly in cancer, with tissue-dependent roles. Additionally, LSD2 extends beyond histone demethylation, impacting DNA methylation, cancer cell reprogramming, E3 ubiquitin ligase activity and DNA damage repair pathways. This study underscores the distinct roles of LSD2, providing insights into their contributions to cancer and other cellular processes.

SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polß Polymerase Activity.

In the cell, DNA polymerase ß (Polß) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polß can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polß variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polß functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.

Combining inotuzumab ozogamicin with PARP inhibitors olaparib and talazoparib exerts synergistic cytotoxicity in acute lymphoblastic leukemia by inhibiting DNA strand break repair.

Inotuzumab ozogamicin (IO), a novel therapeutic drug for relapsed or refractory acute lymphoblastic leukemia (RR)­(ALL), is a humanized anti­cluster of differentiation (CD) 22 monoclonal antibody conjugated with calicheamicin that causes DNA single­ and double­strand breaks. Although the efficacy of IO is significantly improved compared with that of conventional chemotherapies, the prognosis for RR­ALL remains poor, highlighting the need for more effective treatment strategies. The present study examined the role of DNA damage repair inhibition using the poly (ADP­ribose) polymerase (PARP) inhibitors olaparib or talazoparib on the enhancement of the antitumor effects of IO on B­ALL cells in vitro. The Reh, Philadelphia (Ph)­B­ALL and the SUP­B15 Ph+ B­ALL cell lines were used for experiments. Both cell lines were ~90% CD22+. The half­maximal inhibitory concentration (IC50) values of IO were 5.3 and 49.7 ng/ml for Reh and SUP­B15 cells, respectively. The IC50 values of IO combined with minimally toxic concentrations of olaparib or talazoparib were 0.8 and 2.9 ng/ml for Reh cells, respectively, and 36.1 and 39.6 ng/ml for SUP­B15 cells, respectively. The combination index of IO with olaparib and talazoparib were 0.19 and 0.56 for Reh cells and 0.76 and 0.89 for SUP­B15 cells, demonstrating synergistic effects in all combinations. Moreover, the addition of minimally toxic concentrations of PARP inhibitors augmented IO­induced apoptosis. The alkaline comet assay, which quantitates the amount of DNA strand breaks, was used to investigate the degree to which DNA damage observed 1 h after IO administration was repaired 6 h later, reflecting successful repair of DNA strand breaks. However, DNA strand breaks persisted 6 h after IO administration combined with olaparib or talazoparib, suggesting inhibition of the repair processes by PARP inhibitors. Adding olaparib or talazoparib thus synergized the antitumor effects of IO by inhibiting DNA strand break repair via the inhibition of PARP.

Tardigrades: Trained to be hardy in the face of DNA damage.

Tardigrades withstand ionizing irradiation levels ∼500 times higher than humans can tolerate. Two recent papers shed light on how this might be achieved - via the transcriptional induction of DNA repair genes, the induction of a radioprotective DNA-binding protein, and possibly also the heightened capacity of repair proteins.

Molecular and cellular consequences of mitochondrial DNA double-stranded breaks.

Mitochondria are subcellular organelles essential for life. Beyond their role in producing energy, mitochondria govern various physiological mechanisms, encompassing energy generation, metabolic processes, apoptotic events, and immune responses. Mitochondria also contain genetic material that is susceptible to various forms of damage. Mitochondrial double-stranded breaks (DSB) are toxic lesions that the nucleus repairs promptly. Nevertheless, the significance of DSB repair in mammalian mitochondria is controversial. This review presents an updated view of the available research on the consequences of mitochondrial DNA DSB from the molecular to the cellular level. We discuss the crucial function of mitochondrial DNA damage in regulating processes such as senescence, integrated stress response, and innate immunity. Lastly, we discuss the potential role of mitochondrial DNA DSB in mediating the cellular consequences of ionizing radiations, the standard of care in treating solid tumors.

HMGN1 loss sensitizes lung cancer cells to chemotherapy.

The high mobility group nucleosome binding (HMGN) family, constitutes a large family of non-histone protein family known to bind the acidic patch of the nucleosomes with various key cellular functions. Several studies have highlighted the pivotal roles of HMGNs in the pathogenic process of various cancer types. However, the roles of HMGN family in lung adenocarcinoma (LUAD) have not been fully elucidated. Herein, integrative analyses of multiple-omics data revealed that HMGNs frequently exhibit dysregulation in LUAD. Subsequent analysis of the clinical relevance of HMGN1 demonstrated its association with poor prognosis in LUAD and its potential as a diagnostic marker to differentiate LUAD from healthy controls. Additionally, functional enrichment analysis suggested that HMGN1 was mainly involved in DNA repair. To corroborate these findings, cellular experiments were conducted, confirming HMGN1's crucial involvement in homologous recombination repair and its potential to enhance the sensitivity of LUAD cells to standard chemotherapeutic drugs. This study proposes HMGN1 as a novel prognostic biomarker and a promising target for chemotherapy in lung adenocarcinoma.

Iron promotes ovarian cancer malignancy and advances platinum resistance by enhancing DNA repair via FTH1/FTL/POLQ/RAD51 axis.

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of 'Iron addiction', an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

CRISPR-Cas13d screens identify KILR, a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

BACKGROUND: Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. METHODS: We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. RESULTS: We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. CONCLUSIONS: Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

A PARP1-TIMELESS alliance in cancer therapy.

In a recent paper in Nature, Petropoulos et al.1 report that PARP1 acts together with the replisome components TIMELESS and TIPIN to protect the genome from transcription-replication conflicts, which has important implications for the clinical use of PARP inhibitors.

Post-translational modification-dependent oligomerization switch in regulation of global transcription and DNA damage repair during genotoxic stress.

Mechanisms of functional cross-talk between global transcriptional repression and efficient DNA damage repair during genotoxic stress are poorly known. In this study, using human AF9 as representative of Super Elongation Complex (SEC) components, we delineate detailed mechanisms of these processes. Mechanistically, we describe that Poly-Serine domain-mediated oligomerization is pre-requisite for AF9 YEATS domain-mediated TFIID interaction-dependent SEC recruitment at the promoter-proximal region for release of paused RNA polymerase II. Interestingly, during genotoxic stress, CaMKII-mediated phosphorylation-dependent nuclear export of AF9-specific deacetylase HDAC5 enhances concomitant PCAF-mediated acetylation of K339 residue. This causes monomerization of AF9 and reduces TFIID interaction for transcriptional downregulation. Furthermore, the K339 acetylation-dependent enhanced AF9-DNA-PKc interaction leads to phosphorylation at S395 residue which reduces AF9-SEC interaction resulting in transcriptional downregulation and efficient repair of DNA damage. After repair, nuclear re-entry of HDAC5 reduces AF9 acetylation and restores its TFIID and SEC interaction to restart transcription.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

Exploring the Mechanisms of Yishen Tongluo Decoction on Repairing DNA Damage in Mouse Spermatogonia Cells Based on Whole Transcriptome Sequencing.

The aim of this study was to investigate the potential mechanism through which Yishen Tongluo decoction (YSTL) repairs DNA damage caused by benzo(a)pyrene diol epoxide (BPDE) in mouse spermatocytes (GC-2). The GC-2 cells were divided randomly into the control group, BPDE group, and low-, medium-, and high-dose YSTL groups of YSTL decoction. A comet assay was used to detect the DNA fragment index (DFI) of cells in each group. Based on the DFI results, whole transcriptome sequencing was conducted, followed by trend analysis, gene ontology (GO) enrichment analysis, kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and ceRNA network analysis. Compared with the control group, the BPDE group reported a significant increase in the DNA fragmentation index (DFI) (p < .05). Compared with the BPDE group, the low-, high- and medium-dose YSTL groups had a significantly reduced DFI (p < .05). Whole-transcriptome sequencing revealed seven differentially expressed circRNAs, 203 differentially expressed miRNAs, and 3,662 differentially expressed mRNAs between the control group and the BPDE group. There was a total of 12 differentially expressed circRNAs, 204 miRNAs, and 2150 mRNAs between the BPDE group and the traditional Chinese medicine group. The pathways involved include DNA repair pathway, nucleotide excision repair pathway, base excision repair pathway, etc. The ceRNA network reported that Hmga2 was the core protein involved, novel_cir_000117 and mmu-miR-466c-3p were located upstream of Hmga2, and they were regulatory factors associated with Hmga2. Finally, we conclude that YSTL decoction may repair sperm DNA damage caused by BPDE through the novel_cir_000117-mmu-miR-466c-3p-Hmga2 pathway.