Mismatch repair enzymes regulate telomere recombination in Saccharomycescerevisiae.

DNA mismatch repair (MMR) is a crucial mechanism that ensures chromosome stability and prevents the development of various human cancers. Apart from its role in correcting mismatches during DNA replication, MMR also plays a significant role in regulating recombination between non-identical sequences, a process known as homeologous recombination. Telomeres, the protective ends of eukaryotic chromosomes, possess sequences that are not perfectly homologous. While telomerase primarily maintains telomere length in the yeast Saccharomyces cerevisiae, recombination between telomeres becomes a major pathway for length maintenance in cells lacking telomerase. This study investigates the participation of MMR in telomere recombination. Our findings reveal that mutations in MMR genes activate type I recombination. Notably, among the MMR proteins, MutSα (Msh2 and Msh6) and MutLα (Mlh1 and Pms1) exerted the most pronounced effects on telomere recombination. We also found that yeast cells containing simple human telomeric TTAGGG DNA sequences preferentially utilize type II recombination to maintain their telomeres, highlighting the influence of the heterogeneous nature of yeast telomeric sequences on type II recombination. Furthermore, our observations indicate that MMR activity is indispensable for its impact on telomere recombination. Collectively, these results contribute to a more comprehensive understanding of the role of MMR in telomere recombination.

EGFR suppression contributes to growth inhibitory activity of G-quadruplex ligands in non-small cell lung cancers.

Non-small cell lung carcinomas (NSCLCs) commonly harbor activating mutations in the epidermal growth factor receptor (EGFR). Drugs targeting the tyrosine kinase activity of EGFR have shown effectiveness in inhibiting the growth of cancer cells with EGFR mutations. However, the development of additional mutations in cancer cells often leads to the persistence of the disease, necessitating alternative strategies to overcome this challenge. We explored the efficacy of stabilizing the G-quadruplex structure formed in the promoter region of EGFR as a means to suppress its expression and impede the growth of cancer cells with EGFR mutations. We revealed that the carbazole derivative BMVC-8C3O effectively suppressed EGFR expression and demonstrated significant growth inhibition in EGFR-mutated NSCLC cells, both in cell culture and mouse xenograft models. Importantly, the observed repression of EGFR expression and growth inhibition were not exclusive to carbazole derivatives, as several other G-quadruplex ligands exhibited similar effects. The growth-inhibitory activity of BMVC-8C3O is attributed, at least in part, to the repression of EGFR, although it is possible that additional cellular targets are also affected. Remarkably, the growth-inhibitory effect was observed even in osimertinib-resistant cells, indicating that BMVC-8C3O holds promise for treating drug-resistant NSCLC. Our findings present a promising and innovative approach for inhibiting the growth of NSCLC cells with EGFR mutations by effectively suppressing EGFR expression. The demonstrated efficacy of G-quadruplex ligands in this study highlights their potential as candidates for further development in NSCLC therapy.

Suppressing c-FOS expression by G-quadruplex ligands inhibits osimertinib-resistant non-small cell lung cancer.

BACKGROUND: Osimertinib is the first-line therapy for patients with non-small cell lung cancer harboring epidermal growth factor receptor-activating alterations. Although osimertinib has been shown to elicit profound patient responses, cancer cells frequently develop additional alterations that sustain their proliferation capacity. This acquired resistance represents a substantial hurdle in precision medicine for patients with lung cancer. METHODS: The biological and cellular properties of the G-quadruplex ligand BMVC-8C3O and its anticancer activities were evaluated in non-small cell lung carcinomas. In addition, combined treatment with BMVC-8C3O and osimertinib was evaluated for its effects on the growth of osimertinib-resistant tumors in vivo. RESULTS: We demonstrate that BMVC-8C3O effectively suppresses c-FOS expression by stabilizing G-rich sequences located at the c-FOS promoter. The suppression c-FOS expression by BMVC-8C3O increases the sensitivity of acquired resistant cancer cells to osimertinib. Combining BMVC-8C3O and osimertinib has a synergistic effect in inhibiting the growth of acquired resistant cancers both in vitro and in mouse models. The combined inhibitory effect is not limited to BMVC-8C3O, either: several G-quadruplex ligands show varying levels of inhibition activity. We also show that simultaneous inhibition of both the c-FOS and PI3K/AKT pathways by BMVC-8C3O and osimertinib synergistically inhibits the growth of acquired resistant cancer cells. CONCLUSION: These findings unveil a synthetic lethal strategy to prevent and inhibit epidermal growth factor receptor-altered lung cancers with acquired osimertinib resistance. G-quadruplex ligands have the potential to be integrated into current osimertinib-based treatment regimens.

Flap endonuclease Rad27 cleaves the RNA of R-loop structures to suppress telomere recombination.

The long non-coding telomeric RNA transcript TERRA, in the form of an RNA-DNA duplex, regulates telomere recombination. In a screen for nucleases that affects telomere recombination, mutations in DNA2, EXO1, MRE11 and SAE2 cause severe delay in type II survivor formation, indicating that type II telomere recombination is mediated through a mechanism similar to repairing double-strand breaks. On the other hand, mutation in RAD27 results in early formation of type II recombination, suggesting that RAD27 acts as a negative regulator in telomere recombination. RAD27 encodes a flap endonuclease that plays a role in DNA metabolism, including replication, repair and recombination. We demonstrate that Rad27 suppresses the accumulation of the TERRA-associated R-loop and selectively cleaves TERRA of R-loop and double-flapped structures in vitro. Moreover, we show that Rad27 negatively regulates single-stranded C-rich telomeric DNA circles (C-circles) in telomerase-deficient cells, revealing a close correlation between R-loop and C-circles during telomere recombination. These results demonstrate that Rad27 participates in telomere recombination by cleaving TERRA in the context of an R-loop or flapped RNA-DNA duplex, providing mechanistic insight into how Rad27 maintains chromosome stability by restricting the accumulation of the R-loop structure within the genome.

Unveiling a novel serpinB2-tripeptidyl peptidase II signaling axis during senescence.

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and ß-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.

Sequential Phosphorylation of Hepatitis C Virus NS5A Protein Requires the ATP-Binding Domain of NS3 Helicase.

The propagation of the hepatitis C virus (HCV) is regulated in part by the phosphorylation of its nonstructural protein NS5A that undergoes sequential phosphorylation on several highly conserved serine residues and switches from a hypo- to a hyperphosphorylated state. Previous studies have shown that NS5A sequential phosphorylation requires NS3 encoded on the same NS3-NS4A-NS4B-NS5A polyprotein. Subtle mutations in NS3 without affecting its protease activity could affect NS5A phosphorylation. Given the ATPase domain in the NS3 COOH terminus, we tested whether NS3 participates in NS5A phosphorylation similarly to the nucleoside diphosphate kinase-like activity of the rotavirus NSP2 nucleoside triphosphatase (NTPase). Mutations in the NS3 ATP-binding motifs blunted NS5A hyperphosphorylation and phosphorylation at serines 225, 232, and 235, whereas a mutation in the RNA-binding domain did not. The phosphorylation events were not rescued with wild-type NS3 provided in trans. When provided with an NS3 ATPase-compatible ATP analog, N6-benzyl-ATP-γ-S, thiophosphorylated NS5A was detected in the cells expressing the wild-type NS3-NS5B polyprotein. The thiophosphorylation level was lower in the cells expressing NS3-NS5B with a mutation in the NS3 ATP-binding domain. In vitro assays with a synthetic peptide and purified wild-type NS3 followed by dot blotting and mass spectrometry found weak NS5A phosphorylation at serines 222 and 225 that was sensitive to an inhibitor of casein kinase Iα but not helicase. When casein kinase Iα was included in the assay, much stronger phosphorylation was observed at serines 225, 232, and 235. We concluded that NS5A sequential phosphorylation requires the ATP-binding domain of the NS3 helicase and that casein kinase Iα is a potent NS5A kinase. IMPORTANCE For more than 20 years, NS3 was known to participate in NS5A sequential phosphorylation. In the present study, we show for the first time that the ATP-binding domain of NS3 is involved in NS5A phosphorylation. In vitro assays showed that casein kinase Iα is a very potent kinase responsible for NS5A phosphorylation at serines 225, 232, and 235. Our data suggest that ATP binding by NS3 probably results in conformational changes that recruit casein kinase Iα to phosphorylate NS5A, initially at S225 and subsequently at S232 and S235. Our discovery reveals intricate requirements of the structural integrity of NS3 for NS5A hyperphosphorylation and HCV replication.

Dynamic DNA Shortening by Telomere-Binding Protein Cdc13.

Telomeres are essential for chromosome maintenance. Cdc13 is a single-stranded telomeric DNA binding protein that caps telomeres and regulates telomerase function in yeast. Although specific binding of Cdc13 to telomeric DNA is critical for telomere protection, the detail mechanism how Cdc13-DNA complex protects telomere is unclear. Using two single-molecule methods, tethered particle motion and atomic force microscopy, we demonstrate that specific binding of Cdc13 on single-stranded telomeric DNA shortens duplex DNA into distinct states differed by ∼70-80 base pairs. DNA shortening by Cdc13 is dynamic and independent of duplex DNA sequences or length. Significantly, we found that Pif1 helicase is incapable of removing Cdc13 from the shortened DNA-Cdc13 complex, suggesting that Cdc13 forms structurally stable complex by shortening of the bound DNA. Together our data identified shortening of DNA by Cdc13 and provided an indication for efficient protection of telomere ends by the shortened DNA-Cdc13 complex.

CDC25B induces cellular senescence and correlates with tumor suppression in a p53-dependent manner.

The phosphatase cell division cycle 25B (Cdc25B) regulates cell cycle progression. Increased Cdc25B levels are often detected in cancer cell lines and human cancers and have been implicated in contributing to tumor growth, potentially by providing cancer cells with the ability to bypass checkpoint controls. However, the specific mechanism by which increased Cdc25B impacts tumor progression is not clear. Here we analyzed The Cancer Genome Atlas (TCGA) database and found that patients with high CDC25B expression had the expected poor survival. However, we also found that high CDC25B expression had a p53-dependent tumor suppressive effect in lung cancer and possibly several other cancer types. Looking in more detail at the tumor suppressive function of Cdc25B, we found that increased Cdc25B expression caused inhibition of cell growth in human normal fibroblasts. This effect was not due to alteration of specific cell cycle stage or inhibition of apoptosis, nor by induction of the DNA damage response. Instead, increased CDC25B expression led cells into senescence. We also found that p53 was required to induce senescence, which might explain the p53-dependent tumor suppressive function of Cdc25B. Mechanistically, we found that the Cdc25B phosphatase activity was required to induce senescence. Further analysis also found that Cdc25B stabilized p53 through binding and dephosphorylating p53. Together, this study identified a tumor-suppressive function of Cdc25B that is mediated through a p53-dependent senescence pathway.

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress.

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

A Low-Toxicity DNA-Alkylating N-Mustard-Quinoline Conjugate with Preferential Sequence Specificity Exerts Potent Antitumor Activity Against Colorectal Cancer.

Efficacy and safety are fundamental prerequisites for anticancer drug development. In the present study, we explored the anti-colorectal cancer (CRC) activity of SL-1, a DNA-directed N-mustard-quinoline conjugate. The N-mustard moiety in SL-1 induced DNA strand breaks, interstrand cross-links (ICLs), G2/M arrest, and apoptosis, whereas its quinoline moiety preferentially directed SL-1 to target the selective guanine sequence 5'-G-G/C-N-G-C/T-3'. Notably, SL-1 was highly cytotoxic to various CRC cell lines. Experiments using xenograft models revealed that SL-1 was more potent than 5-fluorouracil (5-FU) and oxaliplatin for suppressing the growth of RKO and RKO-E6 (oxaliplatin-resistant subline) cells as well as metastatic SW620 cells. In addition, SL-1 combined with 5-FU was more effective than oxaliplatin and 5-FU for suppressing RKO or SW620 cell growth in mice. Significantly, compared with cisplatin, oxaliplatin, or 5-FU, SL-1 alone or in combination with 5-FU did not cause obvious kidney or liver toxicity in ICR mice. In summary, SL-1, a DNA-directed alkylating agent, is established as an anti-CRC agent with high efficacy and low toxicity and thus warrants further development for the treatment of CRC patients.

The serine protease inhibitor serpinB2 binds and stabilizes p21 in senescent cells.

SerpinB2 is a serine protease inhibitor also known as plasminogen activator inhibitor type 2 (PAI-2). It has been well documented that serpinB2 is an inhibitor of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). Interestingly, serpinB2 levels are increased in senescent cells and serpinB2 is thus considered a senescence biomarker. In this study, by mimicking the elevated levels of serpinB2 in senescent cells, proliferating human fibroblasts were induced into senescence. Senescence induced by serpinB2 did not relate to its extracellular function, as inhibition of serpinB2 secretion, exogenous introduced serpinB2, or a serpinB2 mutant that failed to bind to its extracellular target uPA did not affect senescence. We also showed that serpinB2 is a direct downstream target of p53 that is activated by the DNA damage response pathway. Significantly, serpinB2 bound to and stabilized p21 to mediate senescence in a proteasome-independent manner, indicating that serpinB2 has a direct role in senescence. Thus, this study reveals a unique mechanism by which serpinB2 maintains senescence through stabilization of p21 protein levels.

Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro.

Telomerase is the enzyme involved in extending telomeric DNA. Control of telomerase activity by modulating its access to chromosome ends is one of the most important fundamental mechanisms. This study established an in vitro yeast telomerase reconstitution system that resembles telomere replication in vivo. In this system, a tailed-duplex DNA formed by telomeric DNA was employed to mimic the structure of telomeres. The core catalytic components of telomerase Est2/Tlc1 RNA were used as the telomeric DNA extension machinery. Using the reconstituted systems, this study found that binding of Cdc13 to telomeric DNA inhibited the access of telomerase to its substrate. The result was further confirmed by a single-molecule approach using the tethered-particle motion (TPM)-based telomerase assay. The findings also showed that the inhibitory effect can be relieved by telomerase-associated protein Est1, consistent with the role of Cdc13 and Est1 in regulating telomere extension in vivo. Significantly, this study found that the DNA binding property of Cdc13 was altered by Est1, providing the first mechanistic evidence of Est1 regulating the access of telomerase to its substrate. Thus, the roles of Cdc13 and Est1 in modulating telomerase activity were clearly defined using the in vitro reconstituted system.

Identification of a new class of WNT1 inhibitor: Cancer cells migration, G-quadruplex stabilization and target validation.

Developing the Wnt pathway inhibitors has been considered as a therapeutic approach for cancers and other Wnt-related diseases. Previously we found that the G-rich sequence of WNT1 promoter is capable of forming G-quadruplex structure and stabilizing agents for Wnt1-mediated signaling pathway. Using a established cell-based drug screen system that enabled the evaluation of WNT1 expression activity in a G-quadruplex structure dependent manner, we evaluated a series of 6-substituted 9-chloro-11H-indeno[1,2-c]quinolin-11-one derivatives that potentially inhibit the Wnt1-mediated signaling pathway. The most potent compound SJ26 showed repression of WNT1 activity in a G-quadruplex structure-dependent manner. Moreover, compound SJ26 inhibited the WNT1-mediated downstream signaling pathway and suppressed migration activity of cancer cells. Thus, we have identified a tetracyclic azafluorenone, SJ26, that is capable of binding to G-quadruplex DNA structure, repressing WNT1 expression, and inhibiting cell migration.

Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells. Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target.

A Fluorescent Anti-Cancer Agent, 3,6-bis(1-methyl-4-vinylpyridinium) Carbazole Diiodide, Stains G-Quadruplexes in Cells and Inhibits Tumor Growth.

Compelling evidence suggests that formation of guanine-quadruplex (G4) can protect the integrity of chromosome ends in eukaryotes, and regulate the activity of some gene promoters. In addition, G4 may be a novel therapeutic target. Thus, a number of ligands have been synthesized to stabilize G4. However, skepticism lingers over the existence of G4 in cells, as well as its biological function. The molecule 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) can be used not only as a fluorescent probe to map endogenous and exogenous G4 in live cells, but also as therapeutic agent that arrests cancer growth by inhibiting telomerase activity and regulating gene expression. Thus, the fluorescence of a G4 anti-cancer agent is an invaluable tool to detect G4 in cells, investigate ligand-G4 interaction in live cells, examine the biological function of G4, and guide the development of new fluorescent anti-cancer agents.

The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors.

Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity.

Conformational transition of a hairpin structure to G-quadruplex within the WNT1 gene promoter.

The role of G-quadruplexes (G4s) in biological systems has been widely studied. It is found that they have an important function in gene transcription and regulation. In this work, we have identified two topologies of hairpin and G4 structures formed by a native G-rich sequence (WT22: 5'-GGGCCACCGGGCAGGGGGCGGG-3') from the WNT1 promoter region using nuclear magnetic resonance (NMR) spectroscopy. With the help of site-specific isotope labeling, the topologies of these two structures are unambiguously characterized. Circular dichroism and NMR results are analyzed to determine the kinetics associated with the potassium ion-induced hairpin-to-G4 transition, which is very slow-on the time scale of 4800 s-compared to the previously reported folding kinetics of G4 formation. In addition, the free energies of the unfolding of these two structures are obtained using differential scanning calorimetry. Combining the kinetic and thermodynamic data, we have established the free energy landscape of this two-state folding system. Considering that similar conformational change may exist in other native G-rich sequences, this work highlights an important hairpin to G4 conformational transition which can be used in manipulation of gene regulation or ligand modulation in vivo.

Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends.

Telomerase, a ribonucleoprotein complex, is responsible for maintaining the telomere length at chromosome ends. Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3'-end single-stranded, repetitive telomeric DNA sequence. Pif1, a 5'-to-3' helicase, has been suggested to regulate telomerase activity. We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional extension processes. This telomerase removal efficiency increases at longer ssDNA gaps and at higher Pif1 concentrations. The enhanced telomerase removal efficiency by Pif1 at the longer single-stranded telomeric DNA suggests a way of how Pif1 regulates telomerase activity and maintains telomere length.