Search Results (13)

Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF–ERCC1 nuclease forming a damaged 5′-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5′-flap and different size gap to analyze possible repair factor–replication factor interactions. Ternary XPA–FEN1–DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA–FEN1 complex formation in the absence of DNA due to protein–protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA–XPA–FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA–FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1. Full article

To improve breast cancer treatment and to enable new strategies for therapeutic resistance, therapeutic targets are constantly being studied. Potential targets are proteins of DNA repair and replication and genomic integrity, such as Flap Endonuclease 1 (FEN1). This study investigated the effects of FEN1 inhibitor FEN1-IN-4 in combination with ionizing radiation on cell death, clonogenic survival, the cell cycle, senescence, doubling time, DNA double-strand breaks and micronuclei in breast cancer cells, breast cells and healthy skin fibroblasts. Furthermore, the variation in the baseline FEN1 level and its influence on treatment prognosis was investigated. The cell lines show specific response patterns in the aspects studied and have heterogeneous baseline FEN1 levels. FEN1-IN-4 has cytotoxic, cytostatic and radiosensitizing effects, expressed through increasing cell death by apoptosis and necrosis, G2M share, senescence, double-strand breaks and a reduced survival fraction. Nevertheless, some cells are less affected by the cytotoxicity and fibroblasts show a rather limited response. In vivo, high FEN1 mRNA expression worsens the prognosis of breast cancer patients. Due to the increased expression in breast cancer tissue, FEN1 could represent a new tumor and prognosis marker and FEN1-IN-4 may serve as a new potent agent in personalized medicine and targeted breast cancer therapy. Full article

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. Full article

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3′-5′ exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity. Full article

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication. Full article

DNA damage repair plays a key role in maintaining genomic stability and integrity. Flap endonuclease 1 (FEN1) is a core protein in the base excision repair (BER) pathway and participates in Okazaki fragment maturation during DNA replication. Several studies have implicated FEN1 in the regulation of other DNA repair pathways, including homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Abnormal expression or mutation of FEN1 in cells can cause a series of pathological responses, leading to various diseases, including cancers. Moreover, overexpression of FEN1 contributes to drug resistance in several types of cancers. All this supports the hypothesis that FEN1 could be a therapeutic target for cancer treatment. Targeting FEN1 has been verified as an effective strategy in mono or combined treatment of cancer. Small-molecule compounds targeting FEN1 have also been developed and detected in cancer regression. In this review, we summarize the recent development of small-molecule inhibitors targeting FEN1 in recent years, thereby expanding their therapeutic potential and application. Full article

Target-induced differences in the electrostatic interactions between methylene blue (MB) and indium tin oxide (ITO) electrode surface was firstly employed to develop a homogeneous electrochemical biosensor for flap endonuclease 1 (FEN1) detection. In the absence of FEN1, the positively charged methylene blue (MB) is free in the solution and can diffuse onto the negatively charged ITO electrode surface easily, resulting in an obvious electrochemical signal. Conversely, with the presence of FEN1, a 5′-flap is cleaved from the well-designed flapped dumbbell DNA probe (FDP). The remained DNA fragment forms a closed dumbbell DNA probe to trigger hyperbranched rolling circle amplification (HRCA) reaction, generating plentiful dsDNA sequences. A large amount of MB could be inserted into the produced dsDNA sequences to form MB-dsDNA complexes, which contain a large number of negative charges. Due to the strong electrostatic repulsion between MB-dsDNA complexes and the ITO electrode surface, a significant signal drop occurs. The signal change (ΔCurrent) shows a linear relationship with the logarithm of FEN1 concentration from 0.04 to 80.0 U/L with a low detection limit of 0.003 U/L (S/N = 3). This study provides a label-free and homogeneous electrochemical platform for evaluating FEN1 activity. Full article

Accumulating evidence has suggested a role of the small GTPase Ras homolog gene family member A (RhoA) in DNA damage response (DDR) in addition to its traditional function of regulating cell morphology. In DDR, 2 key components of DNA repair, ataxia telangiectasia-mutated (ATM) and flap structure-specific endonuclease 1 (FEN1), along with intracellular reactive oxygen species (ROS) have been shown to regulate RhoA activation. In addition, Rho-specific guanine exchange factors (GEFs), neuroepithelial transforming gene 1 (Net1) and epithelial cell transforming sequence 2 (Ect2), have specific functions in DDR, and they also participate in Ras-related C3 botulinum toxin substrate 1 (Rac1)/RhoA interaction, a process which is largely unappreciated yet possibly of significance in DDR. Downstream of RhoA, current evidence has highlighted its role in mediating cell cycle arrest, which is an important step in DNA repair. Unraveling the mechanism by which RhoA modulates DDR may provide more insight into DDR itself and may aid in the future development of cancer therapies. Full article

Helicobacter pylori infection induces a plethora of DNA damages. Gastric epithelial cells, in order to maintain genomic integrity, require an integrous DNA damage repair (DDR) machinery, which, however, is reported to be modulated by the infection. CagA is a major H. pylori virulence factor, associated with increased risk for gastric carcinogenesis. Its pathogenic activity is partly regulated by phosphorylation on EPIYA motifs. Our aim was to identify effects of H. pylori infection and CagA on DDR, investigating the transcriptome of AGS cells, infected with wild-type, ΔCagA and EPIYA-phosphorylation-defective strains. Upon RNA-Seq-based transcriptomic analysis, we observed that a notable number of DDR genes were found deregulated during the infection, potentially resulting to base excision repair and mismatch repair compromise and an intricate deregulation of nucleotide excision repair, homologous recombination and non-homologous end-joining. Transcriptome observations were further investigated on the protein expression level, utilizing infections of AGS and GES-1 cells. We observed that CagA contributed to the downregulation of Nth Like DNA Glycosylase 1 (NTHL1), MutY DNA Glycosylase (MUTYH), Flap Structure-Specific Endonuclease 1 (FEN1), RAD51 Recombinase, DNA Polymerase Delta Catalytic Subunit (POLD1), and DNA Ligase 1 (LIG1) and, contrary to transcriptome results, Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) upregulation. Our study accentuates the role of CagA as a significant contributor of H. pylori infection-mediated DDR modulation, potentially disrupting the balance between DNA damage and repair, thus favoring genomic instability and carcinogenesis. Full article

Many early studies presented beneficial effects of polyunsaturated fatty acids (PUFA) on cardiovascular risk factors and disease. However, results from recent meta-analyses indicate that this effect would be very low or nil. One of the factors that may contribute to the inconsistency of the results is that, in most studies, genetic factors have not been taken into consideration. It is known that fatty acid desaturase (FADS) gene cluster in chromosome 11 is a very important determinant of plasma PUFA, and that the prevalence of the single nucleotide polymorphisms (SNPs) varies greatly between populations and may constitute a bias in meta-analyses. Previous genome-wide association studies (GWAS) have been carried out in other populations and none of them have investigated sex and Mediterranean dietary pattern interactions at the genome-wide level. Our aims were to undertake a GWAS to discover the genes most associated with serum PUFA concentrations (omega-3, omega-6, and some fatty acids) in a scarcely studied Mediterranean population with metabolic syndrome, and to explore sex and adherence to Mediterranean diet (MedDiet) interactions at the genome-wide level. Serum PUFA were determined by NMR spectroscopy. We found strong robust associations between various SNPs in the FADS cluster and omega-3 concentrations (top-ranked in the adjusted model: FADS1-rs174547, p = 3.34 × 10⁻¹⁴; FADS1-rs174550, p = 5.35 × 10⁻¹⁴; FADS2-rs1535, p = 5.85 × 10⁻¹⁴; FADS1-rs174546, p = 6.72 × 10⁻¹⁴; FADS2-rs174546, p = 9.75 × 10⁻¹⁴; FADS2-rs174576, p = 1.17 × 10⁻¹³; FADS2-rs174577, p = 1.12 × 10⁻¹², among others). We also detected a genome-wide significant association with other genes in chromosome 11: MYRF (myelin regulatory factor)-rs174535, p = 1.49 × 10⁻¹²; TMEM258 (transmembrane protein 258)-rs102275, p = 2.43 × 10⁻¹²; FEN1 (flap structure-specific endonuclease 1)-rs174538, p = 1.96 × 10⁻¹¹). Similar genome-wide statistically significant results were found for docosahexaenoic fatty acid (DHA). However, no such associations were detected for omega-6 PUFAs or linoleic acid (LA). For total PUFA, we observed a consistent gene*sex interaction with the DNTTIP2 (deoxynucleotidyl transferase terminal interacting protein 2)-rs3747965 p = 1.36 × 10⁻⁸. For adherence to MedDiet, we obtained a relevant interaction with the ME1 (malic enzyme 1) gene (a gene strongly regulated by fat) in determining serum omega-3. The top-ranked SNP for this interaction was ME1-rs3798890 (p = 2.15 × 10⁻⁷). In the regional-wide association study, specifically focused on the FADS1/FASD2/FADS3 and ELOVL (fatty acid elongase) 2/ELOVL 5 regions, we detected several statistically significant associations at p < 0.05. In conclusion, our results confirm a robust role of the FADS cluster on serum PUFA in this population, but the associations vary depending on the PUFA. Moreover, the detection of some sex and diet interactions underlines the need for these associations/interactions to be studied in all specific populations so as to better understand the complex metabolism of PUFA. Full article

This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity. Full article

The human DNA2 (DNA replication helicase/nuclease 2) protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER), interacting with the replication protein A (RPA) and the flap endonuclease 1 (FEN1). DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN) and Bloom syndrome protein (BLM). In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB) repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes. Full article

Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.–441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy. Full article