Chromosomal DNA Replication Research Articles

An integrated view of the structure and function of the human 4D nucleome.

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

Reconstruction of a robust bacterial replication module.

Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes.

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Influence and mechanism of typical transition metal ions on the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria

To investigate the inhibitory effects of various transition metal ions on nitrogen removal and their underlying mechanisms, the single and combined effects of Cu2+ Ni2+ and Zn2+ on Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria Acinetobacter sp. TAC-1 were studied in a batch experiment system. The results revealed that increasing concentrations of Cu2+ and Ni2+ had a detrimental effect on the removal of ammonium nitrogen (NH4+-N) and total nitrogen (TN). Specifically, Cu2+ concentration of 10 mg/L, the TN degradation rate was 55.09%, compared to 77.60% in the control group. Cu2+ exhibited a pronounced inhibitory effect. In contrast, Zn2+ showed no apparent inhibitory effect on NH4+-N removal and even enhanced TN removal at lower concentrations. However, when the mixed ion concentration of Zn2++Ni2+ exceeded 5 mg/L, the removal rates of NH4+-N and TN were significantly reduced. Moreover, transition metal ions did not significantly impact the removal rates of chemical oxygen demand (COD). The inhibition model fitting results indicated that the inhibition sequence was Cu2+ > Zn2+ > Ni2+. Transcriptome analysis demonstrated that metal ions influence TAC-1 activity by modulating the expression of pivotal genes, including zinc ABC transporter substrate binding protein (znuA), ribosomal protein (rpsM), and chromosome replication initiation protein (dnaA) and DNA replication of TAC-1 under metal ion stress, leading to disruptions in transcription, translation, and cell membrane structure. Finally, a conceptual model was proposed by us to summarize the inhibition mechanism and possible response strategies of TAC-1 bacteria under metal ion stress, and to address the lack of understanding regarding the influence mechanism of TAC-1 on nitrogen removal in wastewater co-polluted by metal and ammonia nitrogen. The results provided practical guidance for the management of transition metal and ammonia nitrogen co-polluted water bodies, as well as the removal of high nitrogen.

The budding yeast Fkh1 Forkhead associated (FHA) domain promoted a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Identification of Hub genes with prognostic values in colorectal cancer by integrated bioinformatics analysis.

Our study aimed to investigate the Hub genes and their prognostic value in colorectal cancer (CRC) via bioinformatics analysis. The data set of colorectal cancer was downloaded from the GEO database (GSE21510, GSE110224 and GSE74602) for differential expression analysis using the GEO2R tool. Hub genes were screened by protein-protein interaction (PPI) comprehensive analysis. GEPIA was used to verify the expression of Hub genes and evaluate its prognostic value. The protein expression of Hub gene in CRC was analyzed using the Human Protein Atlas database. The cBioPortal was used to analyze the type and frequency of Hub gene mutations, and the effects of mutation on the patients' prognosis. The TIMER database was used to study the correlation between Hub genes and immune infiltration in CRC. Gene set enrichment analysis (GSEA) was used to explore the biological function and signal pathway of the Hub genes and corresponding co-expressed genes. We identified 346 differentially expressed genes (DEGs), including 117 upregulated and 229 downregulated. Four Hub genes (AURKA, CCNB1, EXO1 and CCNA2) were selected by survival analysis and differential expression validation. The protein and mRNA expression levels of AURKA, CCNB1, EXO1 and CCNA2 were higher in CRC tissues than in adjacent tissues. There were varying degrees of immune cell infiltration and gene mutation of Hub genes, especially B cells and CD8+ T cells. The results of GSEA showed that Hub genes and their co-expressed genes mainly participated in chromosome segregation, DNA replication, translational elongation and cell cycle. Overexpression of AURKA, CCNB1, CCNA2 and EXO1 had a better prognosis for CRC and this effect was correlation with gene mutation and infiltration of immune cells.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA.

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Abstract C154: Investigation of indole-based molecules as a new class of TLK1 inhibitors in prostate cancer therapy

Abstract Targeting protein kinases is an attractive strategy in cancer therapy owing to their importance in cell signaling pathways. Tousled-like kinases (TLKs) are associated with chromosomal integrity, DNA replication, and repair. However, the dysregulation of these genes can give rise to different aberrations. The activity of TLK1, a human isoform of TLK, is found to be attenuated in the case of prostate cancer and breast cancer, as it can phosphorylate many proteins of the DNA Damage Response (DDR) pathway, hence making TLK1 a novel druggable target. To date, phenothiazines-which are known antipsychotic drugs, are the only class of inhibitors reported against TLK1. Our work focuses on developing a new class of TLK1 inhibitors to broaden the spectrum of understanding TLK1 inhibition. As an approach, we designed, synthesized, and validated indole-based molecules with potent TLK1 inhibition via in-silico studies. We further explored the synthesized inhibitors to understand their inhibition against recombinantly purified TLK1 in the presence of its different substrates. Also, the cell viability of these inhibitors was examined against breast cancer and prostate cancer. We found that the inhibitors are more potent in prostate cancer cell lines, as observed by the lowered downstream phosphorylation levels of TLK1 substrates in those cells. The in-vitro, cell, and docking studies imply that the newly designed inhibitors have better potency against TLK1 than its reported inhibitors. We anticipate that our step towards exploring a new class of potent TLK1 inhibitors would aid in elevating the therapeutics in a combinatorial approach to existing prostate cancer therapy.

Centripetal nuclear shape fluctuations associate with chromatin condensation in early prophase

The nucleus plays a central role in several key cellular processes, including chromosome organisation, DNA replication and gene transcription. Recent work suggests an association between nuclear mechanics and cell-cycle progression, but many aspects of this connection remain unexplored. Here, by monitoring nuclear shape fluctuations at different cell cycle stages, we uncover increasing inward fluctuations in late G2 and in early prophase, which are initially transient, but develop into instabilities when approaching the nuclear-envelope breakdown. We demonstrate that such deformations correlate with chromatin condensation by perturbing both the chromatin and the cytoskeletal structures. We propose that the contrasting forces between an extensile stress and centripetal pulling from chromatin condensation could mechanically link chromosome condensation with nuclear-envelope breakdown, two main nuclear processes occurring during mitosis.

Episomal and chromosomal DNA replication and recombination in Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebiasis. DNA replication studies in E. histolytica first started with the ribosomal RNA genes located on episomal circles. Unlike most plasmids, Entamoeba histolytica rDNA circles lacked a fixed origin. Replication initiated from multiple sites on the episome, and these were preferentially used under different growth conditions. In synchronized cells the early origins mapped within the rDNA transcription unit, while at later times an origin in the promoter-proximal upstream intergenic spacer was activated. This is reminiscent of eukaryotic chromosomal replication where multiple potential origins are used. Biochemical studies on replication and recombination proteins in Entamoeba histolytica picked up momentum once the genome sequence was available. Sequence search revealed homologs of DNA replication and recombination proteins, including meiotic genes. The replicative DNA polymerases identified included the α, δ, ε of polymerase family B; lesion repair polymerases Rev1 and Rev3; a translesion repair polymerase of family A, and five families of polymerases related to family B2. Biochemical analysis of EhDNApolA confirmed its polymerase activity with expected kinetic constants. It could perform strand displacement, and translesion synthesis. The purified EhDNApolB2 had polymerase and exonuclease activities, and could efficiently bypass some types of DNA lesions. The single DNA ligase (EhDNAligI) was similar to eukaryotic DNA ligase I. It was a high-fidelity DNA ligase, likely involved in both replication and repair. Its interaction with EhPCNA was also demonstrated. The recombination-related proteins biochemically characterized were EhRad51 and EhDmc1. Both shared the canonical properties of a recombinase and could catalyse strand exchange over long DNA stretches. Presence of Dmc1 indicates the likelihood of meiosis in this parasite. Direct evidence of recombination in Entamoeba histolytica was provided by use of inverted repeat sequences located on plasmids or chromosomes. In response to a variety of stress conditions, and during encystation in Entamoeba invadens, recombination-related genes were upregulated and homologous recombination was enhanced. These data suggest that homologous recombination could have critical roles in trophozoite growth and stage conversion. Availability of biochemically characterized replication and recombination proteins is an important resource for exploration of novel anti-amoebic drug targets.

Bacterial Argonaute Proteins Aid Cell Division in the Presence of Topoisomerase Inhibitors in Escherichia coli.

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix rosea (LrAgo) are active in heterologous Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin, depending on the host double-strand break repair machinery. Both pAgos are preferentially loaded with small guide DNAs (smDNAs) derived from the sites of replication termination. Ciprofloxacin increases the amounts of smDNAs from the termination region and from the sites of genomic DNA cleavage by gyrase, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin enhances asymmetry in the distribution of smDNAs around Chi sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases may help to complete replication of chromosomal DNA by promoting chromosome decatenation or participating in the processing of gyrase cleavage sites, and may switch their functional activities depending on the host species. IMPORTANCE Prokaryotic Argonautes (pAgos) are programmable nucleases with incompletely understood functions in vivo. In contrast to eukaryotic Argonautes, most studied pAgos recognize DNA targets. Recent studies suggested that pAgos can protect bacteria from invader DNA and counteract phage infection and may also have other functions including possible roles in DNA replication, repair, and gene regulation. Here, we have demonstrated that two cyanobacterial pAgos, SeAgo and LrAgo, can assist DNA replication and facilitate cell division in the presence of topoisomerase inhibitors in Escherichia coli. They are specifically loaded with small guide DNAs from the region of replication termination and protect the cells from the action of the gyrase inhibitor ciprofloxacin, suggesting that they help to complete DNA replication and/or repair gyrase-induced breaks. The results show that pAgo proteins may serve as a backup to topoisomerases under conditions unfavorable for DNA replication and may modulate the resistance of host bacterial strains to antibiotics.

Bacterial DNA methyltransferase: A key to the epigenetic world with lessons learned from proteobacteria.

Epigenetics modulates expression levels of various important genes in both prokaryotes and eukaryotes. These epigenetic traits are heritable without any change in genetic DNA sequences. DNA methylation is a universal mechanism of epigenetic regulation in all kingdoms of life. In bacteria, DNA methylation is the main form of epigenetic regulation and plays important roles in affecting clinically relevant phenotypes, such as virulence, host colonization, sporulation, biofilm formation et al. In this review, we survey bacterial epigenomic studies and focus on the recent developments in the structure, function, and mechanism of several highly conserved bacterial DNA methylases. These methyltransferases are relatively common in bacteria and participate in the regulation of gene expression and chromosomal DNA replication and repair control. Recent advances in sequencing techniques capable of detecting methylation signals have enabled the characterization of genome-wide epigenetic regulation. With their involvement in critical cellular processes, these highly conserved DNA methyltransferases may emerge as promising targets for developing novel epigenetic inhibitors for biomedical applications.

Juvenile zebrafish (Danio rerio) are able to recover from lordosis

Haemal lordosis, a frequent skeletal deformity in teleost fish, has long been correlated with increased mechanical loads induced by swimming activity. In the present study, we examine whether juvenile zebrafish can recover from haemal lordosis and explore the musculoskeletal mechanisms involved. Juveniles were subjected to a swimming challenge test (SCT) that induced severe haemal lordosis in 49% of the animals and then immediately transferred them to 0.0 total body lengths (TL) per second of water velocity for a week. The recovery from lordosis was examined by means of whole mount staining, histology and gene expression analysis. Results demonstrate that 80% of the lordotic zebrafish are capable of internal and external recovery within a week after the SCT. Recovered individuals presented normal shape of the vertebral centra, maintaining though distorted internal tissue organization. Through the transcriptomic analysis of the affected haemal regions, several processes related to chromosome organization, DNA replication, circadian clock and transcription regulation were enriched within genes significantly regulated behind this musculoskeletal recovery procedure. Genes especially involved in adipogenesis, bone remodeling and muscular regeneration were regulated. A remodeling tissue-repair hypothesis behind haemal lordosis recovery is raised. Limitations and future possibilities for zebrafish as a model organism to clarify mechanically driven musculoskeletal changes are discussed.

615 yRNA4 up-regulation induces collagen expression in scleroderma cultured dermal fibroblasts

yRNA is an essential factor for chromosomal DNA replication and has a functional role. Four yRNA subtypes have shown some diagnostic and prognostic potential for several types of cancers and diseases. The exact etiology of systematic sclerosis (SSc) requires clarification, and there are no biomarkers to measure the severity of fibrosis caused by accumulation of collagen. We therefore investigated the role of yRNAs in the pathogenesis of collagen accumulation in SSc. First, we investigated the expression of yRNAs in total RNA extracted from SSc and normal skin tissues by real-time PCR.

359P Analysis of TMB and tumor microenvironment in Polymerase epsilon (POLE) deficient colorectal cancer

POLE (DNA Polymerase Epsilon, Catalytic Subunit) is a protein coding gene. This gene encodes the catalytic subunit of DNA polymerase epsilon, involved in DNA repair and chromosomal DNA replication. Mutations in this gene, especially related to proofreading defects in the POLE polymerase (POLE pd), have been associated with hypermutated genomes and sensitive response by checkpoint blocking immunotherapies for colorectal cancer (CRC). The profile of Tumor mutation burden (TMB) and tumor microenvironment between POLE mutation(POLEmut) and POLE wildtype(POLEwt) CRC is not clearly elaborated.

SUMOylation in Skeletal Development, Homeostasis, and Disease.

The modification of proteins by small ubiquitin-related modifier (SUMO) molecules, SUMOylation, is a key post-translational modification involved in a variety of biological processes, such as chromosome organization, DNA replication and repair, transcription, nuclear transport, and cell signaling transduction. In recent years, emerging evidence has shown that SUMOylation regulates the development and homeostasis of the skeletal system, with its dysregulation causing skeletal diseases, suggesting that SUMOylation pathways may serve as a promising therapeutic target. In this review, we summarize the current understanding of the molecular mechanisms by which SUMOylation pathways regulate skeletal cells in physiological and disease contexts.

The mechanism of replication stalling and recovery within repetitive DNA

Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.

RAE1 is a prognostic biomarker and is correlated with clinicopathological characteristics of patients with hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a primary malignant tumor that accounts for approximately 90% of all cases of primary liver cancer worldwide. Microtubule alterations may contribute to the broad spectrum of resistance to chemotherapy, tumor development, and cell survival. This study aimed to assess the value of ribonucleic acid export 1 (RAE1), as a regulator of microtubules, in the diagnosis and prognosis of HCC, and to analyze its correlation with genetic mutations and pathways in HCC.ResultsThe mRNA and protein levels of RAE1 were significantly elevated in HCC tissues compared with those in normal tissues. The high expression level of RAE1 was correlated with T stage, pathologic stage, tumor status, histologic grade, and alpha-fetoprotein level. HCC patients with a higher expression level of RAE1 had a poorer prognosis, and the expression level of RAE1 showed the ability to accurately distinguish tumor tissues from normal tissues (area under the curve (AUC) = 0.951). The AUC values of 1-, 3-, and 5-year survival rates were all above 0.6. The multivariate Cox regression analysis showed that RAE1 expression level was an independent prognostic factor for a shorter overall survival of HCC patients. The rate of RAE1 genetic alterations was 1.1% in HCC samples. Gene ontology and kyoto encyclopedia of genes and genomes pathway enrichment analyses indicated the co-expressed genes of RAE1 were mainly related to chromosome segregation, DNA replication, and cell cycle checkpoint. Protein–protein interaction analysis showed that RAE1 was closely correlated with NUP205, NUP155, NUP214, NUP54, and NXF1, all playing important roles in cell division and mitotic checkpoint.ConclusionRAE1 can be a potential diagnostic and prognostic biomarker associated with microtubules and a therapeutic target for HCC.