Replication Arrest Research Articles

Beneficial and detrimental genes in the cellular response to replication arrest.

DNA replication is essential for all living organisms. Several events can disrupt replication, including DNA damage (e.g., pyrimidine dimers, crosslinking) and so-called "roadblocks" (e.g., DNA-binding proteins or transcription). Bacteria have several well-characterized mechanisms for repairing damaged DNA and then restoring functional replication forks. However, little is known about the repair of stalled or arrested replication forks in the absence of chemical alterations to DNA. Using a library of random transposon insertions in Bacillus subtilis, we identified 35 genes that affect the ability of cells to survive exposure to an inhibitor that arrests replication elongation, but does not cause chemical alteration of the DNA. Genes identified include those involved in iron-sulfur homeostasis, cell envelope biogenesis, and DNA repair and recombination. In B. subtilis, and many bacteria, two nucleases (AddAB and RecJ) are involved in early steps in repairing replication forks arrested by chemical damage to DNA and loss of either nuclease causes increased sensitivity to DNA damaging agents. These nucleases resect DNA ends, leading to assembly of the recombinase RecA onto the single-stranded DNA. Notably, we found that disruption of recJ increased survival of cells following replication arrest, indicating that in the absence of chemical damage to DNA, RecJ is detrimental to survival. In contrast, and as expected, disruption of addA decreased survival of cells following replication arrest, indicating that AddA promotes survival. The different phenotypes of addA and recJ mutants appeared to be due to differences in assembly of RecA onto DNA. RecJ appeared to promote too much assembly of RecA filaments. Our results indicate that in the absence of chemical damage to DNA, RecA is dispensable for cells to survive replication arrest and that the stable RecA nucleofilaments favored by the RecJ pathway may lead to cell death by preventing proper processing of the arrested replication fork.

Displacement and dissociation of oligonucleotides during DNA hairpin closure under strain.

The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus significantly reducing its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbour dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) of various lengths in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in numerous biological and bioengineering contexts.

Mechanism of action of non-camptothecin inhibitor Genz-644282 in topoisomerase I inhibition

Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT), causes replication arrest and cell death. Although CPT has excellent cytotoxicity, it has the disadvantage of instability under physiological conditions. Therefore, new types of TOP1 inhibitor have attracted particular attention. Here, we characterised the effect of a non-camptothecin inhibitor, Genz-644282 (Genz). First, we found that treatment with Genz showed cytotoxicity by introducing double-strand breaks (DSBs), which was suppressed by co-treatment with aphidicolin. Genz-induced DSB formation required the functions of TOP1. Next, we explored the advantages of Genz over CPT and found it was effective against CPT-resistant TOP1 carrying either N722S or N722A mutation. The effect of Genz was also confirmed at the cellular level using a CPT-resistant cell line carrying N722S mutation in the TOP1 gene. Moreover, we found arginine residue 364 plays a crucial role for the binding of Genz. Because tyrosine residue 723 is the active centre for DNA cleavage and re-ligation by TOP1, asparagine residue 722 plays crucial roles in the accessibility of the drug. Here, we discuss the mechanism of action of Genz on TOP1 inhibition.

Both phosphorylation and phosphatase activity of PTEN are required to prevent replication fork progression during stress by inducing heterochromatin

PTEN is a tumor suppressor protein frequently altered in various cancers. PTEN-null cells have a characteristic of rapid proliferation with an unstable genome. Replication stress is one of the causes of the accumulation of genomic instability if not sensed by the cellular signaling. Though PTEN-null cells have shown to be impaired in replication progression and stalled fork recovery, the association between the catalytic function of PTEN regulated by posttranslational modulation and cellular response to replication stress has not been studied explicitly. To understand molecular mechanism, we find that PTEN-null cells display unrestrained replication fork progression with accumulation of damaged DNA after treatment with aphidicolin which can be rescued by ectopic expression of full-length PTEN, as evident from DNA fiber assay. Moreover, the C-terminal phosphorylation (Ser 380, Thr 382/383) of PTEN is essential for its chromatin association and sensing replication stress that, in response, induce cell cycle arrest. Further, we observed that PTEN induces HP1α expression and H3K9me3 foci formation in a C-terminal phosphorylation-dependent manner. However, phosphatase dead PTEN cannot sense replication stress though it can be associated with chromatin. Together, our results suggest that DNA replication perturbation by aphidicolin enables chromatin association of PTEN through C-terminal phosphorylation, induces heterochromatin formation by stabilizing and up-regulating H3K9me3 foci and augments CHK1 activation. Thereby, PTEN prevents DNA replication fork elongation and simultaneously causes G1-S phase cell cycle arrest to limit cell proliferation in stress conditions. Thus PTEN act as stress sensing protein during replication arrest to maintain genomic stability.

The Senescence Markers p16INK4A, p14ARF/p19ARF, and p21 in Organ Development and Homeostasis.

It is widely accepted that senescent cells accumulate with aging. They are characterized by replicative arrest and the release of a myriad of factors commonly called the senescence-associated secretory phenotype. Despite the replicative cell cycle arrest, these cells are metabolically active and functional. The release of SASP factors is mostly thought to cause tissue dysfunction and to induce senescence in surrounding cells. As major markers for aging and senescence, p16INK4, p14ARF/p19ARF, and p21 are established. Importantly, senescence is also implicated in development, cancer, and tissue homeostasis. While many markers of senescence have been identified, none are able to unambiguously identify all senescent cells. However, increased levels of the cyclin-dependent kinase inhibitors p16INK4A and p21 are often used to identify cells with senescence-associated phenotypes. We review here the knowledge of senescence, p16INK4A, p14ARF/p19ARF, and p21 in embryonic and postnatal development and potential functions in pathophysiology and homeostasis. The establishment of senolytic therapies with the ultimate goal to improve healthy aging requires care and detailed knowledge about the involvement of senescence and senescence-associated proteins in developmental processes and homeostatic mechanism. The review contributes to these topics, summarizes open questions, and provides some directions for future research.

DIPG-46. Radiation induced senescence in diffuse intrinsic pontine glioma cells reveals selective vulnerability to Bcl-XL inhibition

Diffuse intrinsic pontine glioma remains a devastating condition with a dismal five year survival rate less than 5%. New approaches for treating this aggressive disease are critical to driving progress. Conventional radiotherapy remains the cornerstone of treatment, with no chemotherapeutic agent found to improve survival. However, radiotherapy is often delivered as a palliative treatment, and disease often recurs 3-6 months after. Radiation causes DNA damage and oxidative stress yielding a senescent state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumour recurrence. Targeted ablation of non-replicating senescent tumour cells following radiation could negate tumour recurrence. It remains unknown whether DIPG undergoes senescence following radiation, and furthermore, whether senolytics can be utilised to target senescent DIPG cells. We employed radiation to induce a senescent state in primary human DIPG cell lines. Senescence was confirmed using SA-β-gal staining, lack of EdU incorporation and qRT-PCR to characterise the SASP in three primary human DIPG cell lines. RNA-sequencing on DIPG cells following radiation revealed senescence and SASP signatures. Likewise, expression of senescence markers has been detected in human tumours. Viable cells that survive radiation were then utilised to screen candidate senolytic drugs, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiation treated DIPG cells. In addition, Bcl-XL degradation using PROTACs (proteolysis targeting chimeras) resulted in a significant increase in senolysis of susceptible tumour cells. Conversely, Bcl-2 inhibitors failed to show any consistent senolytic activity. We are currently performing preclinical studies in the mouse to test the efficiency of senolytics against DIPG. These results demonstrate future possibilities of targeting radiation induced senescence in DIPG, using novel senolytic therapies and highlight Bcl-XL dependency as a potential vulnerability of surviving DIPG cells following exposure to radiation.

Fibroblast Senescence: A Risk Factor for Remodeling, Inflammation, and Arrhythmias in the Post‐MI Heart

ObjectivesCellular senescence is a stress response involving permanent replicative arrest. It is accompanied by a complex senescence‐associated secretory phenotype (SASP), which is characterized by the presence of pro‐inflammatory cytokines and chemokines, growth factors, and tissue‐remodeling metalloproteinases. Little is known regarding the role of senescence and the SASP in the context of arrhythmias post myocardial infarction (MI). Here, we characterized the arrhythmogenic cardiac consequences and molecular mechanisms underlying tissue remodeling changes in impaired or accelerated cardiac myofibroblast (CMF) senescence and the pro‐inflammatory SASP during post‐MI wound healing process in genetically engineered mice.Material‐MethodsImpaired or accelerated CMF‐specific senescence was achieved by using cre‐lox mouse models to delete p53 or MDM2 genes. MI surgery via direct ligation of the left anterior descending artery (LAD) was performed in 4‐month‐old wild‐type (WT), Pstn‐Cre(+/‐) p53(fl/fl), Pstn‐MerCreMer(+/‐) MDM2(fl/fl), and respective littermate control (LMC) mice of both sexes. Changes in cardiac structure and function post‐MI were assessed by echocardiography. Senescence‐associated β‐galactosidase (SA‐βgal) staining was applied to detect and quantify senescent cells in three zones: infarct zone (IZ), infarct border zone (IBZ), and remote zone (RZ). Immunofluorescence staining identified the types of senescent cells. Masson’s trichrome staining was used for fibrosis assessment. RT‐qPCR was performed to quantitate the expression of known senescence genes and canonical SASP factors.ResultsPstn‐Cre(+/‐) p53(fl/fl) mice displayed significantly decreased CMF senescence two weeks post‐MI in the scar compared to littermate control p53(fl/fl) and WT mice. We noticed a similar trend in the IBZ, but this did not reach statistical significance. Pstn‐MCM(+/‐) MDM2(fl/fl) mice showed significantly increased CMF senescence in the IZ compared littermate control MDM2(fl/fl) mice. We observed a similar trend in the IBZ, but this did not reach statistical significance. In all mice, we observed essentially no senescence in the RZ. Our data did not show any significant difference in fibrosis between any genotype in any zone. We did not observe any overall trend in differential expression in senescence and SASP genes between p53(fl/fl) and Pstn‐Cre(+/‐) p53(fl/fl) mice in any zone. There was no evidence of any sex‐specific differences in aforementioned results.ConclusionsA significant increase and persistence of CMF senescence likely contributes to pathological tissue remodeling during the post‐MI wound healing process. This is likely due to SASP‐induced pro‐arrhythmogenic chronic inflammation or via direct cell‐cell interactions between senescent CMFs and myocytes.

Physiological and metabolic effects of glyphosate as the sole P source on a cosmopolitan phytoplankter and biogeochemical implications

Nutrient conditions influence the physiology and stoichiometry of marine phytoplankton. While extensive studies have documented the effects of abundances and types of nutrients such as nitrogen (N) and phosphorus (P), the effect of phosphonates as a P source is less understood and underexplored. Here, with the cosmopolitan coccolithophorid Emiliania huxleyi as a model phytoplankter, we investigated the effect of the phosphonate type of herbicide glyphosate as the sole P source in comparison with the P-depleted and P-replete (with 36 μM dissolved inorganic phosphate [DIP]) cultures. We measured changes in cellular C (carbon):P and N:P ratios and physiological performance and documented the corresponding transcriptomic and miRNAomic responses in E. huxleyi to glyphosate treatment. We found that glyphosate supported population growth but not to the full scale relative to DIP, and this was under the concerted regulation of DNA replication and cell cycle arrest genes as well as the growth-regulating miRNA. Furthermore, our data suggest that E. huxleyi took up glyphosate directly, bypassing extracellular hydrolysis, and this involved ABC transporters. Meanwhile, glyphosate-grown cultures displayed marked increases in cellular particulate organic C (POC) and PON contents, cell size, and transcription of genes for CO2 fixation and citrate cycle, nitrate transport, and protein biosynthesis. However, compared to DIP, the maximum absorption rate of glyphosate was only 33%, and glyphosate-grown E. huxleyi cells exhibited a mild P-stress symptom and elevated cellular C:P and N:P ratios. Interestingly, glyphosate-grown cells showed an increased sinking rate, suggesting that glyphosate as the sole P source might enhance the efficiency of C export by E. huxleyi, which would compensate for the expected decline in primary productivity (and hence carbon efflux) in the future more nutrient-depleted ocean. This biogeochemical implication needs to be further studied and verified, however.

Reassembling a cannon in the DNA defense arsenal: Genetics of StySA, a BREX phage exclusion system in Salmonella lab strains.

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX (BacteRiophage EXclusion) systems. These RM-like activities employ host protection by DNA modification, but immediate replication arrest occurs without evident of nuclease action on unmodified phage DNA. Here we show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a variant BREX system. A laboratory strain disabled for both the restriction and methylation activity of StySA nevertheless has wild type sequence in pglX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (μ) in their C-terminal domains. We further investigate this system in situ, replacing the mutated pglZμ and brxCμ genes with the WT counterpart. PglZ-WT supports methylation in the presence of either BrxCμ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. These results suggests that while BrxC, PglZ and PglX are principal components of the BREX modification activity, BrxL is required for restriction only. Furthermore, we show that a partial disruption of brxL disrupts transcription globally.

A spotlight on non-host resistance to plant viruses.

Plant viruses encounter a range of host defenses including non-host resistance (NHR), leading to the arrest of virus replication and movement in plants. Viruses have limited host ranges, and adaptation to a new host is an atypical phenomenon. The entire genotypes of plant species which are imperceptive to every single isolate of a genetically variable virus species are described as non-hosts. NHR is the non-specific resistance manifested by an innately immune non-host due to pre-existing and inducible defense responses, which cannot be evaded by yet-to-be adapted plant viruses. NHR-to-plant viruses are widespread, but the phenotypic variation is often not detectable within plant species. Therefore, molecular and genetic mechanisms of NHR need to be systematically studied to enable exploitation in crop protection. This article comprehensively describes the possible mechanisms of NHR against plant viruses. Also, the previous definition of NHR to plant viruses is insufficient, and the main aim of this article is to sensitize plant pathologists to the existence of NHR to plant viruses and to highlight the need for immediate and elaborate research in this area.

Selective Vulnerability of Senescent Glioblastoma Cells to BCL-XL Inhibition.

Glioblastoma (GBM) is a rapidly fatal malignancy typically treated with radiation and temozolomide (TMZ), an alkylating chemotherapeutic. These cytotoxic therapies cause oxidative stress and DNA damage, yielding a senescent-like state of replicative arrest in surviving tumor cells. Unfortunately, recurrence is inevitable and may be driven by surviving tumor cells eventually escaping senescence. A growing number of so-called “senolytic” drugs have been recently identified that are defined by their ability to selectively eliminate senescent cells. A growing inventory of senolytic drugs is under consideration for several diseases associated with aging, inflammation, DNA damage, as well as cancer. Ablation of senescent tumor cells after radiation and chemotherapy could help mitigate recurrence by decreasing the burden of residual tumor cells at risk of recurrence. This strategy has not been previously explored for GBM. We evaluated a panel of 10 previously described senolytic drugs to determine whether any could exhibit selective activity against human GBM persisting after exposure to radiation or TMZ. Three of the 10 drugs have known activity against BCL-XL and preferentially induced apoptosis in radiated or TMZ-treated glioma. This senolytic activity was observed in 12 of 12 human GBM cell lines. Efficacy could not be replicated with BCL-2 inhibition or senolytic agents acting against other putative senolytic targets. Knockdown of BCL-XL decreased survival of radiated GBM cells, whereas knockdown of BCL-2 or BCL-W yielded no senolytic effect.

DNA Damage Tolerance Pathways in Human Cells: A Potential Therapeutic Target.

DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template. The two main DDT pathways are translesion synthesis (TLS), which involves the recruitment of specialized TLS polymerases to the site of replication arrest to bypass lesions, and homology-directed damage tolerance, which includes the template switching and fork reversal pathways. With some exceptions, lesion bypass by TLS polymerases is a source of mutagenesis, potentially contributing to the development of cancer. The capacity of TLS polymerases to bypass replication-blocking lesions induced by anti-cancer drugs such as cisplatin can also contribute to tumor chemoresistance. On the other hand, during homology-directed DDT the nascent sister strand is transiently utilised as a template for replication, allowing for error-free lesion bypass. Given the role of DNA damage tolerance pathways in replication, mutagenesis and chemoresistance, a more complete understanding of these pathways can provide avenues for therapeutic exploitation. A number of small molecule inhibitors of TLS polymerase activity have been identified that show synergy with conventional chemotherapeutic agents in killing cancer cells. In this review, we will summarize the major DDT pathways, explore the relationship between damage tolerance and carcinogenesis, and discuss the potential of targeting TLS polymerases as a therapeutic approach.

Abstract 10732: Systemic Administration of Telomerase RNA Reverses Vascular Senescence and Extends Lifespan in Progeria Mice

Introduction: Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS, a mutation in lamin A (progerin) alters nuclear morphology and gene expression. iPSCs derived endothelium from progeria patients show impairment in their morphology and function including replication arrest, DNA damage, increased expression of inflammatory markers, and telomere erosion, compared to iPSCs derived endothelial cells from their unaffected parents. We have shown that hTERT mRNA therapy rescues their senescent phenotypes and restores normal function and gene expression in-vitro. To validate our finding in-vivo, we have used lentivirus harboring mouse telomerase to treat mice model of progeria and investigated the beneficial effects on mice vasculature in different organs. Hypothesis: Gene therapy with mRNA encoding mice telomerase improves survival and reversing cell senescence in a mice model of HGPS. Methods: HGPS mice were injected twice with a lentivirus construct encoding mice telomerase via tail vein. Mice were monitored for survival; endothelial cells were isolated at the time of euthanasia and concentrations of cytokines, chemokines, and growth factors were analyzed using Elisa, PCR and immunofluorescence staining. For clinical translation we developed a lipid nanoparticle formulation to deliver mRNA encoding mTERT to mice, validating its function using mRNA encoding Luciferase to demonstrate biodistribution and safety profile. Results: Two treatments with lentiviral mTERT increased the lifespan of HGPS mice by 20% and reduced mediators of inflammation and thrombosis in HGPS such as thrombomodulin. HGPS mice treated with mTERT had lower DNA damage markers and increased SIRT1 in their vasculature and other organs. A Lipid nanoparticle encapsulating mTERT RNA and shows a favorable safety profile and the capacity to systemically and safely deliver luciferase mRNA, generating active luciferase, documenting a predominantly hepatic biodistribution. Conclusions: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases. Further development in systemic delivery is thus underway

Abstract 11821: Telomerase Therapy Reverses Vascular Senescence and Extends Lifespan in Progeria Mice

Background: Hutchinson-Gilford Progeria Syndrome (HGPS) is an accelerated aging syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin), alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, DNA damage, increased expression of inflammatory markers and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular aging. Methods: We generated ECs from iPSCs belong to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. Results: hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acLDL uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects which may be mediated in part by a reduction in progerin expression and an increase in SIRT1. Progeria mice treated with mTERT Lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. Conclusion: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.

MICAL2 Promotes Proliferation and Migration of Glioblastoma Cells Through TGF-β/p-Smad2/EMT-Like Signaling Pathway.

Recent studies showed that molecule interacting with CasL2 (MICAL2) could be a novel tumor growth factor, and it is closely associated with tumor growth and invasion. However, the role it plays in glioblastoma (GBM) and its potential mechanisms are currently unknown. Our study is designed to identify the effect of MICAL2 on GBM cells and the potential mechanisms behind it. Here, we found that MICAL2 interacts with TGF receptor-type I (TGFRI) and promotes the proliferation and migration of glioblastoma through the TGF-β/p-Smad2/EMT-like signaling pathway. MICAL2-knockdown inhibited the proliferation of glioblastoma cells, which was related to cell cycle arrest and downregulation of DNA replication. The invasion abilities of U87 and U251 cells were reduced after the knockdown of MICAL2. MICAL2 promoted the growth of GBM in nude mice. High MICAL2 predicts poor outcome of GBM patients. MICAL2 could be identified as a novel promising therapeutic target for human GBM.

Testis transcriptome profiling identified lncRNAs involved in spermatogenic arrest of cattleyak

Cattleyaks are the crossbred offspring between cattle and yaks, exhibiting the prominent adaptability to the harsh environment as yaks and much higher growth performances than yaks around Qinghai-Tibet plateau. Unfortunately, cattleyak cannot be effectively used in yak breeding due to its male infertility resulted from spermatogenic arrest. In this study, we performed RNA sequencing (RNA-seq) and bioinformatics analysis to determine the expression profiles of long noncoding RNA (lncRNA) from cattleyak and yak testis. A total of 604 differentially expressed (DE) lncRNAs (135 upregulated and 469 downregulated) were identified in cattleyak with respect to yak. Through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we identified several DE lncRNAs regulating the mitotic cell cycle processes by targeting the genes significantly associated with the mitotic cell cycle checkpoint and DNA damage checkpoint term and also significantly involved in p53 signaling pathway, mismatch repair and homologous recombination pathway (P < 0.05). The reverse transcription PCR (RT-PCR) and quantitative Real-Time PCR (qRT-PCR) analysis of the randomly selected fourteen DE lncRNAs and the seven target genes validated the RNA-seq data and their true expressions during spermatogenesis in vivo. Molecular cloning and sequencing indicated that the testis lncRNAs NONBTAT012170 and NONBTAT010258 presented higher similarity among different cattleyak and yak individuals. The downregulation of these target genes in cattleyak contributed to the abnormal DNA replication and spermatogenic arrest during the S phase of mitotic cell cycle. This study provided a novel insight into lncRNA expression profile changes associated with spermatogenic arrest of cattleyak.

Bacteriophage Twort protein Gp168 is a β-clamp inhibitor by occupying the DNA sliding channel.

Bacterial chromosome replication is mainly catalyzed by DNA polymerase III, whose beta subunits enable rapid processive DNA replication. Enabled by the clamp-loading complex, the two beta subunits form a ring-like clamp around DNA and keep the polymerase sliding along. Given the essential role of β-clamp, its inhibitors have been explored for antibacterial purposes. Similarly, β-clamp is an ideal target for bacteriophages to shut off host DNA synthesis during host takeover. The Gp168 protein of phage Twort is such an example, which binds to the β-clamp of Staphylococcus aureus and prevents it from loading onto DNA causing replication arrest. Here, we report a cryo-EM structure of the clamp–Gp168 complex at 3.2-Å resolution. In the structure of the complex, the Gp168 dimer occupies the DNA sliding channel of β-clamp and blocks its loading onto DNA, which represents a new inhibitory mechanism against β-clamp function. Interestingly, the key residues responsible for this interaction on the β-clamp are well conserved among bacteria. We therefore demonstrate that Gp168 is potentially a cross-species β-clamp inhibitor, as it forms complex with the Bacillus subtilis β-clamp. Our findings reveal an alternative mechanism for bacteriophages to inhibit β-clamp and provide a new strategy to combat bacterial drug resistance.

NAD+ bioavailability mediates PARG inhibition-induced replication arrest, intra S-phase checkpoint and apoptosis in glioma stem cells.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

Oleanolic acid blocks the purine salvage pathway for cancer therapy by inactivating SOD1 and stimulating lysosomal proteolysis

Metabolic reprogramming is a core hallmark of cancer and is key for tumorigenesis and tumor progression. Investigation of metabolic perturbation by anti-cancer compounds would allow a thorough understanding of the underlying mechanisms of these agents and identification of new anti-cancer targets. Here, we demonstrated that the administration of oleanolic acid (OA) rapidly altered cancer metabolism, particularly suppressing the purine salvage pathway (PSP). PSP restoration significantly opposed OA-induced DNA replication and cell proliferation arrest, underscoring the importance of this pathway for the anti-cancer activity of OA. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and 5′-nucleotidase (5′-NT), two metabolic enzymes essential for PSP activity, were promptly degraded by OA via the lysosome pathway. Mechanistically, OA selectively targeted superoxide dismutase 1 (SOD1) and yielded reactive oxygen species (ROS) to activate the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/macroautophagy pathway, thus eliciting lysosomal degradation of HGPRT and 5′-NT. Furthermore, we found that the PSP was overactivated in human lung and breast cancers, with a negative correlation with patient survival. The results of this study elucidated a new anti-cancer mechanism of OA by restraining the PSP via the SOD1/ROS/AMPK/mTORC1/macroautophagy/lysosomal pathway. We also identified the PSP as a new target for cancer treatment and highlighted OA as a potential therapeutic agent for cancers with high PSP activity.

Telomerase therapy reverses vascular senescence and extends lifespan in progeria mice.

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated ageing syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin) alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, increased expression of inflammatory markers, DNA damage, and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular ageing. We generated ECs from iPSCs belonging to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage, and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acetylated low-density lipoprotein uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects that may be mediated in part by a reduction in progerin expression and an increase in sirtuin 1 (SIRT1). Progeria mice treated with mTERT lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.