Structure-specific Endonuclease Research Articles

Abstract 2040: Targeting MUS81 endonuclease to enhance chemo-immunotherapy response in head and neck squamous cell carcinoma

Abstract Background: In advanced head and neck squamous cell carcinoma (HNSCC), immune checkpoint inhibitors are only effective in 15-20% of cases. Defects in replication stress response represent a promising avenue for enhancing the therapeutic efficacy of immunotherapy through DNA damage and the release of immunostimulatory DNA. MUS81, a DNA structure-specific endonuclease involved in resolving replication stress, is upregulated in many cancer types including HNSCC, and MUS81-associated cytosolic DNA is implicated in triggering cGAS-STING mediated innate immunity. The objectives of this study were to assess the impact of targeting MUS81 on HNSCC cell sensitivity to cisplatin, and investigate its role in enhancing immunotherapy response in HNSCC, through the cell viability and cell cycle analysis, differential gene expression, and DNA release within small extracellular vesicles (exosomes). Methods and Results: We used HNSCC cell lines and patient-derived organoids (PDOs) to investigate the consequences of CRISPR/Cas9-mediated MUS81 knockout (KO). We demonstrated that MUS81 KO increased HNSCC cell sensitivity to cisplatin, inducing a G2/M cell cycle arrest. Transcriptomic analysis of differentially expressed genes (DEG) in MUS81 KO vs control cells highlighted upregulation of ANXA6 (tumor suppressor gene encoding for Annexin A6), MACROH2A2 (involved in the quiescent phenotype acquisition of malignant HNSCC cells) and CDA (encoding for cytidine deaminase, implicated in replication stress). MUS81 KO was also associated with downregulation of LCP1 (encoding for lymphocyte cytosolic protein 1, upregulated in oral cancer and controlling cellular proliferation, invasiveness, and migratory activities). Gene Set Enrichment Analysis (GSEA) of RNA-seq data, revealed positive enrichment in MUS81 KO cells treated with cisplatin in genes involved in the hallmark interferon alpha (Normalized Enrichment Score (NES) 3.03, p-value < 0.001) and interferon gamma (NES 2.83, p- value < 0.001) pathways, indicative of innate and adaptive immune responses. Additionally, the pathway of hallmark inflammatory response was positively enriched (NES 2.22, p < 0.001). In addition, cisplatin treatment in MUS81 KO was associated with notable alterations in the concentration and length of exosomal DNA, with implications for immune response modulation within the tumor microenvironment. Conclusions: Our study underscores the potential significance of MUS81 as a novel therapeutic target in HNSCC. In addition to sensitizing HNSCC cells to chemotherapeutic agents, targeting MUS81 may influence the dynamics of the tumor microenvironment, thereby enhancing the prospects of immunotherapy in this context. Further in-depth investigations are imperative to validate these observations and facilitate the development of innovative therapeutic strategies tailored to targeting MUS81 in HNSCC. Citation Format: Ali Abdulnabi Suwaidan, Anna Pasto, Caitlin McCarthy, Teng Pan, Jinhai Deng, Luigi Dolcetti, Giovanna Alfano, Rami Mustapha, James Monypenny, Martin Forster, Tony Ng. Targeting MUS81 endonuclease to enhance chemo-immunotherapy response in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2040.

ERCC4: a potential regulatory factor in inflammatory bowel disease and inflammation-associated colorectal cancer.

The pathogenesis of inflammatory bowel disease (IBD) remains unclear and is associated with an increased risk of developing colitis-associated cancer (CAC). Under sustained inflammatory stimulation in the intestines, loss of early DNA damage response genes can lead to tumor formation. Many proteins are involved in the pathways of DNA damage response and play critical roles in protecting genes from various potential damages that DNA may undergo. ERCC4 is a structure-specific endonuclease that participates in the nucleotide excision repair (NER) pathway. The catalytic site of ERCC4 determines the activity of NER and is an indispensable gene in the NER pathway. ERCC4 may be involved in the imbalanced process of DNA damage and repair in IBD-related inflammation and CAC. This article primarily reviews the function of ERCC4 in the DNA repair pathway and discusses its potential role in the processes of IBD-related inflammation and carcinogenesis. Finally, we explore how this knowledge may open novel avenues for the treatment of IBD and IBD-related cancer.

Structure-specific DNA endonuclease T7 endonuclease I cleaves DNA containing UV-induced DNA lesions.

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs, and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication, and repair in E.coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analyzed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD), and 6-4 photoproducts (6-4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5' to the lesion of CPD on the lesion strand. However, in the case of 6-4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5' to the adenine-adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6-4PP. These findings suggest that T7 endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6-4PP.

Noise Stress Abrogates Structure-Specific Endonucleases within the Mammalian Inner Ear.

Nucleotide excision repair (NER) is a multistep biochemical process that maintains the integrity of the genome. Unlike other mechanisms that maintain genomic integrity, NER is distinguished by two irreversible nucleolytic events that are executed by the xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group F (XPF) structure-specific endonucleases. Beyond nucleolysis, XPG and XPF regulate the overall efficiency of NER through various protein-protein interactions. The current experiments evaluated whether an environmental stressor could negatively affect the expression of Xpg (Ercc5: excision repair cross-complementing 5) or Xpf (Ercc4: excision repair cross-complementing 4) in the mammalian cochlea. Ubiquitous background noise was used as an environmental stressor. Gene expression levels for Xpg and Xpf were quantified from the cochlear neurosensory epithelium after noise exposure. Further, nonlinear cochlear signal processing was investigated as a functional consequence of changes in endonuclease expression levels. Exposure to stressful background noise abrogated the expression of both Xpg and Xpf, and these effects were associated with pathological nonlinear signal processing from receptor cells within the mammalian inner ear. Given that exposure to environmental sounds (noise, music, etc.) is ubiquitous in daily life, sound-induced limitations to structure-specific endonucleases might represent an overlooked genomic threat.

Purification of Saccharomyces cerevisiae recombinant Crp1

A complex Mus81-Mm4 is a DNA structure–specific endonuclease in Saccharomyces cerevisiae. Mus81-Mms4 functions in processing of recombination intermediates that could arise during the repair of stalled and blocked replication forks and double stranded breaks. Mus81-Mms4 works with many proteins involved in DNA repair, replication fork stability, and joint molecule formation/resolution during homologous recombination repair. A biochemical screening of protein(s) that enhances the Mus81-Mms4 endonuclease activity on its preferable substrates in vitro revealed that Crp1, a cruciform DNA-recognizing protein, which can specifically bind to DNA four-way junction structures like Holliday junctions could be the potential factor. To further demonstrate that Crp1 interacts functionally with Mus81-Mms4 in vitro, we carried out the purification of recombinant Crp1 using Escherichia coli system. Our results showed that the purified Crp1 was highly homogenous and active that is ready for biochemical use.

Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.

FEN1 inhibitor SC13 promotes CAR-T cells infiltration into solid tumours through cGAS-STING signalling pathway.

It is well known that chimeric antigen receptor T-cell immunotherapy (CAR-T-cell immunotherapy) has excellent therapeutic effect in haematological tumours, but it still faces great challenges in solid tumours, including inefficient T-cell tumour infiltration and poor functional persistence. Flap structure-specific endonuclease 1 (FEN1), highly expressed in a variety of cancer cells, plays an important role in both DNA replication and repair. Previous studies have reported that FEN1 inhibition is an effective strategy for cancer treatment. Therefore, we hypothesized whether FEN1 inhibitors combined with CAR-T-cell immunotherapy would have a stronger killing effect on solid tumours. The results showed that low dose of FEN1 inhibitors SC13 could induce an increase of double-stranded broken DNA (dsDNA) in the cytoplasm. Cytosolic dsDNA can activate the cyclic GMP-AMP synthase-stimulator of interferon gene signalling pathway and increase the secretion of chemokines. In vivo, under the action of FEN1 inhibitor SC13, more chemokines were produced at solid tumour sites, which promoted the infiltration of CAR-T cells and improved anti-tumour immunity. These findings suggest that FEN1 inhibitors could enable CAR-T cells to overcome poor T-cell infiltration and improve the treatment of solid tumours.

Inhibition of FEN1 promotes DNA damage and enhances chemotherapeutic response in prostate cancer cells.

Prostate cancer (PCa) refers to epithelial malignancies occurring in prostate and is the most commonly diagnosed cancer among men. Flap structure-specific endonuclease 1 (FEN1) is one of the major base excise repair enzymes and is abnormally expressed in a variety of cancers, which contributes to cancer progression. Targeting FEN1 serves as a potent strategy for cancer therapy. However, how FEN1 acts on PCa cell proliferation and its role in chemotherapeutic response remain largely unknown. In this study, we show that knockdown of FEN1 by CRISPR/Cas9 system impedes the proliferation and migration of PCa cells. FEN1 Inhibitor SC13 induced DNA damage accumulation and further resulted in apoptosis of PCa cells. Furthermore, genetic knockdown of FEN1 or inhibition of FEN1 by SC13 promoted DNA damage and enhanced docetaxel (DTX)-induced chemotherapeutic response in PCa cells. Collectively, these findings demonstrate the importance of FEN1 in PCa cell proliferation and implicate FEN1 as a promising target for monotherapy or combination therapeutic strategy in PCa treatment.

Identification of small-molecule inhibitors of human MUS81-EME1/2 by FRET-based high-throughput screening

The MUS81-EME1/2 structure-specific endonucleases play a crucial role in the processing of stalled replication forks and recombination intermediates, and have been recognized as an attractive drug target to potentiate the anti-cancer efficacy of DNA-damaging agents. Currently, no bioactive small-molecule inhibitors of MUS81 are available. Here, we performed a high-throughput small-molecule inhibitors screening, using the FRET-based DNA cleavage assay. From 7920 compounds, we identified dyngo-4a as a potent inhibitor of MUS81 complexes. Dyngo-4a effectively inhibits the endonuclease activities of both MUS81-EME1 and MUS81-EME2 complexes, with IC50 values of 0.57 μM and 2.90 μM, respectively. Surface plasmon resonance (SPR) and electrophoretic mobility shift assay (EMSA) assays reveal that dyngo-4a directly binds to MUS81 complexes (KD ∼ 0.61 μM) and prevents them from binding to DNA substrates. In HeLa cells, dyngo-4a significantly suppresses bleomycin-triggered H2AX serine 139 phosphorylation (γH2AX). Together, our results demonstrate that dyngo-4a is a potent MUS81 inhibitor, which could be further developed as a potentially valuable chemical tool to explore more physiological roles of MUS81 in the cells.

Explore the dominant factor in prime editing via a view of DNA processing

Prime editing is a revolutionary gene-editing method that is capable of introducing insertions, deletions and base substitutions into the genome. However, the editing efficiency of Prime Editor (PE) is limited by the DNA repair process. Here, we show that overexpression of the flap structure-specific endonuclease 1 (FEN1) and the DNA ligase 1 (LIG1) increases the efficiency of prime editing, which is similar to the dominant negative mutL homolog 1 (MLH1dn). In addition, MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing. Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.

MUS81 cleaves TOP1-derived lesions and other DNA–protein cross-links

BackgroundDNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs.ResultsThis study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation.ConclusionsOur data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

Downregulation of MUS81 expression inhibits cell migration and maintains EBV latent infection through miR-BART9-5p in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) infection is associated with the occurrence and development of gastric cancer (GC). Methyl methanesulfonate and ultraviolet-sensitive gene 81 (MUS81) is the catalytic component of a structure-specific endonuclease and plays an important role in chromosomal stability. However, the link between EBV infection and MUS81 remains unclear. In the present study, we found that MUS81 expression was much lower in EBV-associated GC cells than in EBV-negative GC. MUS81 acts as an oncogene in GC by inducing the cell migration and proliferation. Western blot and luciferase reporter assays revealed that miR-BART9-5p directly targeted MUS81 and downregulated its expression. Additionally, overexpression of MUS81 in EBV-positive GC cells inhibited the expression of EBV nuclear antigen 1 (EBNA1). EBNA1 is critical for the pathogenesis of EBV-associated tumors and the maintenance of a stable copy number of the viral genomes. Altogether, these results indicated that the lowering MUS81 expression might be a mechanism by EBV to maintain its latent infection.

Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Fluorescently labeled oligonucleotides are powerful tools for characterizing DNA processes; however, their use is limited by the cost and sequence requirements of current labeling technologies. Here, we develop an easy, inexpensive, and sequence-independent method for site-specifically labeling DNA oligonucleotides. We utilize commercially synthesized oligonucleotides containing phosphorothioate diester(s) in which a nonbridging oxygen is replaced with a sulfur (PS-DNA). The increased nucleophilicity of the thiophosphoryl sulfur relative to the phosphoryl oxygen permits selective reactivity with iodoacetamide compounds. As such, we leverage a long-existing bifunctional linker, N,N′-bis(α-iodoacetyl)-2-2′-dithiobis(ethylamine) (BIDBE), that reacts with PS-DNAs to leave a free thiol, allowing conjugation of the wide variety of commercial maleimide-functionalized compounds. We optimized BIDBE synthesis and its attachment to PS-DNA and then fluorescently labeled the BIDBE-PS-DNA using standard protocols for labeling cysteines. We purified the individual epimers, and using single-molecule Förster resonance energy transfer (FRET), we show that the FRET efficiency is independent of the epimeric attachment. Subsequently, we demonstrate that an epimeric mixture of double-labeled Holliday junctions (HJs) can be used to characterize their conformational properties in the absence and presence of the structure-specific endonuclease Drosophila melanogaster Gen. Finally, we use a biochemical activity assay to show that this double-labeled HJ is functional for cleavage by Gen and that the double-labeled HJ allows multiple DNA species to be identified in a single experiment. In conclusion, our results indicate that dye-labeled BIDBE-PS-DNAs are comparable to commercially labeled DNAs at a significantly reduced cost. Notably, this technology could be applied to other maleimide-functionalized compounds, such as spin labels, biotin, and proteins. The sequence independence of labeling, coupled with its ease and low cost, enables unrestricted exploration of dye placement and choice, providing the potential for creation of differentially labeled DNA libraries and opening previously inaccessible experimental avenues.

Helicase activities of Rad5 and Rrm3 genetically interact in the prevention of recombinogenic DNA lesions in Saccharomyces cerevisiae

The genome must be monitored to ensure its duplication is completed accurately to prevent genome instability. In Saccharomyces cerevisiae, the 5′ to 3′ DNA helicase Rrm3, a member of the conserved PIF1 family, facilitates replication fork progression through an unknown mechanism. Disruption of Rrm3 helicase activity leads to increased replication fork pausing throughout the yeast genome. Here, we show that Rrm3 contributes to replication stress tolerance in the absence of the fork reversal activity of Rad5, defined by its HIRAN domain and DNA helicase activity, but not in the absence of Rad5′s ubiquitin ligase activity. The Rrm3 and Rad5 helicase activities also interact in the prevention of recombinogenic DNA lesions, and DNA lesions that accumulate in their absence need to be salvaged by a Rad59-dependent recombination pathway. Disruption of the structure-specific endonuclease Mus81 leads to accumulation of recombinogenic DNA lesions and chromosomal rearrangements in the absence of Rrm3, but not Rad5. Thus, at least two mechanisms exist to overcome fork stalling at replication barriers, defined by Rad5-mediated fork reversal and Mus81-mediated cleavage, and contribute to the maintenance of chromosome stability in the absence of Rrm3.

Localization and expression of SLX4 in the testis of sterile male cattle-yak.

Cattle-yak, the hybrid offspring of yak (Bos grunniens) and cattle (Bos taurus), serves as a unique model to dissect the molecular mechanisms underlying reproductive isolation. While female cattle-yaks are fertile, the males are completely sterile due to spermatogenic arrest at the meiosis stage and massive germ cell apoptosis. Interestingly, meiotic defects are partially rescued in the testes of backcrossed offspring. The genetic basis of meiotic defects in male cattle-yak remains unclear. Structure-specific endonuclease subunit (SLX4) participates in meiotic double-strand break (DSB) formation in mice, and its deletion results in defects in spermatogenesis. In the present study, we examined the expression patterns of SLX4 in the testes of yak, cattle-yak, and backcrossed offspring to investigate its potential roles in hybrid sterility. The results showed that the relative abundances of SLX4 mRNA and protein were significantly reduced in the testis of cattle-yak. The results of immunohistochemistry revealed that SLX4 was predominately expressed in spermatogonia and spermatocytes. Chromosome spreading experiments showed that SLX4 was significantly decreased in the pachytene spermatocytes of cattle-yak compared with yak and backcrossed offspring. These findings suggest that SLX4 expression was dysregulated in the testis of cattle-yak, potentially resulting in the failure of crossover formation and collapses of meiosis in hybrid males.

Identification of potential biomarkers for colorectal cancer by clinical database analysis and Kaplan-Meier curves analysis.

This study aimed to explore critical genes as potential biomarkers for the diagnosis and prognosis of colorectal cancer (CRC) for clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded microarray datasets GSE89076, GSE73360, and GSE32323 from the GEO database identified differentially expressed genes (DEGs), and performed a functional enrichment analysis. A protein-protein interaction network was constructed, and correlated module analysis was performed using STRING and Cytoscape. The Kaplan-Meier survival curve shows the survival of the hub genes. The expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), and PCNA in tissues and changes in tumor grade were analyzed. A total of 329 DEGs were identified, including 264 upregulated and 65 downregulated genes. The functions and pathways of DEGs include the mitotic cell cycle, poly(A) RNA binding replication, ATP binding, DNA replication, ribosome biogenesis in eukaryotes, and RNA transport. Forty-seven Hub genes were identified, and biological process analysis showed that these genes were mainly enriched in cell cycle and DNA replication. Patients with mutations in CDK1, PCNA, and CCNB1 had poorer survival rates. CDK1, PCNA, and CCNB1 were significantly overexpressed in the tumor tissues. The expression of CDK1 and CCNB1 gradually decreased with increasing tumor grade. CDK1, CCNB1, and PCNA can be used as potential markers for the diagnosis and prognosis of CRC. These genes are overexpressed in colon cancer tissues and are associated with low survival rates in CRC patients.

SMC5/6 complex-mediated SUMOylation stimulates DNA-protein cross-link repair in Arabidopsis.

DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.

Causal genetic regulation of DNA replication on immune microenvironment in colorectal tumorigenesis: Evidenced by an integrated approach of trans-omics and GWAS.

The interplay between DNA replication stress and immune microenvironment alterations is known to play a crucial role in colorectal tumorigenesis, but a comprehensive understanding of their association with and relevant biomarkers involved in colorectal tumorigenesis is lacking. To address this gap, we conducted a study aiming to investigate this association and identify relevant biomarkers. We analyzed transcriptomic and proteomic profiles of 904 colorectal tumor tissues and 342 normal tissues to examine pathway enrichment, biological activity, and the immune microenvironment. Additionally, we evaluated genetic effects of single variants and genes on colorectal cancer susceptibility using data from genome-wide association studies (GWASs) involving both East Asian (7062 cases and 195745 controls) and European (24476 cases and 23073 controls) populations. We employed mediation analysis to infer the causal pathway, and applied multiplex immunofluorescence to visualize colocalized biomarkers in colorectal tumors and immune cells. Our findings revealed that both DNA replication activity and the flap structure-specific endonuclease 1 ( FEN1) gene were significantly enriched in colorectal tumor tissues, compared with normal tissues. Moreover, a genetic variant rs4246215 G>T in FEN1 was associated with a decreased risk of colorectal cancer (odds ratio = 0.94, 95% confidence interval: 0.90-0.97, P meta = 4.70 × 10 -9). Importantly, we identified basophils and eosinophils that both exhibited a significantly decreased infiltration in colorectal tumors, and were regulated by rs4246215 through causal pathways involving both FEN1 and DNA replication. In conclusion, this trans-omics incorporating GWAS data provides insights into a plausible pathway connecting DNA replication and immunity, expanding biological knowledge of colorectal tumorigenesis and therapeutic targets.

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.

The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U μL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.