Flap Structure-specific Endonuclease Research Articles

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

Flap Structure-Specific Endonuclease 1 (FEN1) May be a Key Mediator of Genome Instability in Myeloma: A Cellular Vulnerability with Potential Therapeutic Significance

Multiple myeloma is associated with a marked genomic instability which leads to acquisition of mutational changes, some of which underlie disease progression including development of drug resistance and poor clinical outcome. Understanding mechanisms of genomic instability is, therefore, extremely important to develop novel improved therapeutic strategies. Since dysregulated nuclease activity can induce DNA breaks and genetic recombination eventually disrupting genomic integrity, we have evaluated nuclease activity and specific nucleases for their role in genomic instability in MM. We previously identified a nuclease gene signature correlating with genomic instability in a myeloma patient dataset and tested it for correlation with survival in two other datasets. We showed that expression of these genes associated with poor overall as well as event free survival in both datasets, IFM172 (P< 0.00005) and gse24080 (P< 0.0008). We have now further refined this signature to a nine gene signature and tested it for correlation with survival in three different MM patient datasets in which gene expression was evaluated either by microarray (GSE39754, n=170; gse24080; n=559) or RNASeq (n=300). Elevated expression of nine gene signature significantly correlated with poor overall survival in all three datasets (P ≤ 1e-06 for IFM70 and gse24080 and P = 0.002 for RNASeq). To biologically and molecularly validate this signature, we conducted an shRNA screen and evaluated impact of all nine genes in signature as well as four additional nucleases on homologous recombination (HR) activity, using a plasmid based assay in which HR produces a functional luciferase gene. Of thirteen nucleases tested, knockdown of seven was associated with ≥50% inhibition of HR activity; the strongest (~80%) inhibition of HR activity was observed by FEN1 knockdown. To further investigate FEN1, we confirmed the role of this nuclease in HR using a different (DRGFP) assay in which homology-based recombination between two mutated genes, generates a functional GFP gene. Using this assay, we showed that FEN1-knockdown in U2OS cells was also associated with a strong (71%) inhibition of HR activity, confirming the role of this nuclease in dysregulation of HR. Evaluation by Western blotting in three different normal PBMC samples and eleven MM cell lines showed that FEN1 was not detected in normal cells, whereas highly expressed in MM cells. Expression profile using microarray also showed that FEN1 is elevated in a subset of MM patient samples. Knockdown of FEN1 in two MM cell lines, RPMI and H929, led to reduction in overall nuclease activity (by ~40%) as assessed by a fluorescence based nuclease activity assay and a similar (~50%) reduction in the levels of gamma-H2AX, a marker of DNA breaks. These data indicate that FEN1 nuclease activity contributes to increased DNA breaks as well as elevated HR activity in MM cells. To further understand the role of FEN1 in dysregulated HR and genome stability in MM, using mass spectrometry, we have identified the interacting proteins. Role of FEN1 in acquisition of new genomic changes over time in MM cells is cuurently being investigated in our laboratory. In summary, we show that FEN1 is an important component of machinery maintaining genomic integrity and plays a significant role in genome dysregulation in myeloma. The FEN1 dysfunction may provide a cellular vulnerability that can be therapeutically exploited. DisclosuresNo relevant conflicts of interest to declare.

Investigation of human flap structure-specific endonuclease 1 (FEN1) activity on primer-template models and exploration of a substrate-based FEN1 inhibitor

Flap structure-specific endonuclease 1 (FEN1) is one of the enzymes that involve in Eukaryotic DNA replication and repair. Recent studies have proved that FEN1 is highly over-expressed in various types of cancer cells and is a drug target. However, a limited number of FEN1 inhibitors has been identified and approved. Herein, we investigate the catalytic activity of FEN1, and propose a substrate-based inhibitor. As a consequence, one of the phosphorothioate-modified substrates is proved to exhibit the most efficient inhibitory effect in our in vitro examinations. A novelly-designed substrate-based FEN1 inhibitor was accordingly constructed and determined a remarkable IC50 value.

Expression profiling of metastatic renal cell carcinoma using gene set enrichment analysis

To identify complex changes in cell biology occurring during metastatic progression of renal cell carcinoma using a novel gene expression analysis algorithm. Whole genome expression profiling was carried out on 32 snap-frozen samples of clear-cell renal cell carcinoma metastases, 29 primary tumors (14 low grade, 15 high grade) and 14 samples of normal kidney tissue using oligonucleotide microarrays. These data were analyzed with the gene set enrichment analysis method, which is able to detect even small, but significant, expression changes in functionally connected genes that cannot be shown by gene-by-gene comparisons. There were 95 gene sets (pathways) with significant upregulation in metastases compared with normal kidney tissue (P < 0.01), and 77 gene sets with significant downregulation, respectively. Low-grade and high-grade tumors showed deregulation of various pathways that have previously not been described in renal cell carcinoma. There were significant changes of genes involved in cell cycle control, apoptosis, cell motility, metabolism, cell adhesion and cytoskeleton. Some promising new potential therapy targets were identified in renal cell carcinoma metastases; for example, aurora-kinase A and flap structure-specific endonuclease 1. Expression profiling of metastatic renal cell carcinoma using the gene set enrichment analysis pathway analysis method provides new and detailed insights in alterations occurring in renal cell carcinoma during malignant transformation and progression. These data can help to develop new and specifically targeted renal cell carcinoma therapies.

FEN1 (flap structure-specific endonuclease 1)

Review on FEN1 (flap structure-specific endonuclease 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2-3-fold and the other 4-5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 ( Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA -2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.

CaPeptides: Selectively targeting caPCNA and sensitizing gemcitabine in pancreatic cancer cells

Abstract caPeptides that block the functions of caPCNA, cancer associated proliferating cell nuclear antigen in malignant cells has been developed by our laboratory and showed promising anti-cancer therapeutic activities. Previously, we have identified caPCNA, an acidic isoform of PCNA that presents in various cancer cells and appears to be associated, at least in part, with malignant transformation of cells. Since PCNA is involved in DNA replication and repair in prokaryote and eukaryote cells, we hypothesize that caPCNA is likely to perform similar functions specifically in cancer cells. Antibodies developed against caPCNA showed the inhibition of DNA replication in cancer cells. The investigation of the interaction of caPCNA with its binding partners including flap structure-specific endonuclease 1(Fen-1) and xeroderma pimentosum complementation group G (XPG) has led us to identify an amino acid domain sequence that confers the cancer cell specificity of caPCNA. We synthesized caPeptides, small caPCNA related peptides derived from this sequence to promote cancer cell cytotoxicity. caPeptides have the potential ability to compete with several cellular proteins that participate in DNA replication, repair, cell cycle control, transcription, and chromosomal recombination for their naturally occurring binding site on caPCNA and block binding of full length caPCNA to these proteins. The disruption of the interaction between caPCNA and its binding proteins prevents cancer cellular functions (vital cellular machinery) that recruit caPCNA and therefore may lead caPeptides to be cytotoxic by themselves or in combination with cancer chemotherapeutic drugs. Our results demonstrated that these peptides were cytotoxic to pancreatic cancer cells and more significantly they rendered resistant pancreatic cells to be 10-fold more sensitive to gemcitabine, a standard clinical chemotherapeutic agent for treating pancreatic cancer patients. Considering that pancreatic cancer is well known for its high mortality rate due to its chemotherapy ineffectiveness and resistances to gemcitabine our studies render further investigations on the anti-neoplastic action and mechanisms of the peptides in pancreatic cancer.

Abstract 1597: Expression of caPCNA and antineoplastic action of caPeptides in pancreatic cancer cells

Abstract Our laboratory previously reported the identification of an acidic isoform of proliferating cell nuclear antigen (caPCNA) in various cancer cells, which appears to be associated, at least in part, with malignant transformation of cells. The current studies show the expression of caPCNA in BXPC-3, Paca-2 and Capan-1 pancreatic cancer cells. Since Proliferating Cell Nuclear Antigen (PCNA) is involved in DNA replication and repair in prokaryote and eukaryote cells, we hypothesize that caPCNA is likely to perform similar functions specifically in cancer cells. Antibodies developed against caPCNA showed growth inhibition activity in cancer cells suggesting caPCNA is related to the proliferation of malignant cells. In order to begin to understand the function of caPCNA in cellular malignant transformation, we have investigated the interaction of caPCNA with its binding partners including flap structure-specific endonuclease 1(Fen-1) and xeroderma pimentosum complementation group G (XPG). We have identified an amino acid domain sequence that confers the cancer cell specificity of caPCNA. In the course of our studies we synthesized small caPCNA related peptides (caPeptides) derived from this sequence that promote cancer cell cytotoxicity. caPeptides have the potential ability to compete with several cellular proteins that participate in DNA replication, repair, cell cycle control, transcription, and chromosomal recombination for their naturally occurring binding site on caPCNA and block binding of full length caPCNA to these proteins. The disruption of the interaction between caPCNA and its binding proteins prevents cancer cellular functions (vital cellular machinery) that recruit caPCNA and therefore may lead caPCNA-derived peptides to be cytotoxic by themselves or in combination with cancer chemotherapeutic drugs. These peptides, either alone or in combination with other anticancer agents are potentially useful cancer chemotherapeutics or augmentors of the pharmacodynamic effect of specific anti-cancer chemotherapeutics. Our results show that these peptides demonstrate promising growth inhibition and cytotoxicity in pancreatic cancer cells. Considering that pancreatic cancer is well known for its high mortality rate due to its chemotherapy ineffectiveness our studies render further investigations on the action of caPCNA and the anti-neoplastic mechanism of the peptides in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1597.

Identification of flap structure-specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning.

We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.

Wheat (Triticum vulgare) chloroplast nuclease ChSI exhibits 5' flap structure-specific endonuclease activity.

The structure-specific ChSI nuclease from wheat (Triticum vulgare) chloroplast stroma has been previously purified and characterized in our laboratory. It is a single-strand-specific DNA and RNA endonuclease. Although the enzyme has been initially characterized and used as a structural probe, its biological function is still unknown. Localization of the ChSI enzyme inside chloroplasts, possessing their own DNA that is generally highly exposed to UV light and often affected by numerous redox reactions and electron transfer processes, might suggest, however, that this enzyme could be involved in DNA repair. The repair of some types of DNA damage has been shown to proceed through branched DNA intermediates which are substrates for the structure-specific DNA endonucleases. Thus we tested the substrate specificity of ChSI endonuclease toward various branched DNAs containing 5' flap, 5' pseudoflap, 3' pseudoflap, or single-stranded bulged structural motifs. It appears that ChSI has a high 5' flap structure-specific endonucleolytic activity. The catalytic efficiency (k(cat)/K(M)) of the enzyme is significantly higher for the 5' flap substrate than for single-stranded DNA. The ChSI 5' flap activity was inhibited by high concentrations of Mg(2+), Mn(2+), Zn(2+), or Ca(2+). However, low concentrations of divalent cations could restore the loss of ChSI activity as a consequence of EDTA pretreatment. In contrast to other known 5' flap nucleases, the chloroplast enzyme ChSI does not possess any 5'-->3' exonuclease activity on double-stranded DNA. Therefore, we conclude that ChSI is a 5' flap structure-specific endonuclease with nucleolytic activity toward single-stranded substrates.

04/01418 Projecting performance of reintroduced direct sunlight based on the local meteorological features: Kim, G. and Kim, J. T. Solar Energy Materials and Solar Cells, 2003, 80, (1), 85–94

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.

The effects of 1alpha,25-dihydroxyvitamin D3 on the expression of DNA replication genes.

To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3-E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F-driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT-PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3. 1Alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] has a potent antiproliferative action characterized by a blocked transition from the G1- to the S-phase of the cell cycle. This study aims to identify genes whose expression is markedly altered after 1,25(OH)2D3 treatment in parallel with or preceding the observed G1-arrest. The cDNA microarray technique was used, and the expression of approximately 4600 genes in MC3T3-E1 mouse osteoblasts was studied 6 and 12 h after treatment with 10(-8) M 1,25(OH)2D3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed on MC3T3-E1 cells and on wildtype and vitamin D receptor (VDR) knockout primary murine epidermal keratinocytes (VDRwt MEKs, VDR-/- MEKs) and murine mammary tumor cells (GR) to confirm the microarray data. After 12 h of treatment, in parallel with the 1,25(OH)2D3-induced G1 arrest, a particular set of DNA replication genes including a cell division cycle 6 homolog, a DNA polymerase alpha subunit, proliferating cell nuclear antigen, two DNA polymerase delta subunits, and flap-structure specific endonuclease 1, was downregulated at least 2-fold. These genes are known targets of the E2F family of transcription factors, which are probably the central mediators of this action of 1,25(OH)2D3. Indeed, as shown by transfection assays with an E2F reporter construct, 12- and 24-h treatment of MC3T3-E1 cells with 1,25(OH)2D3 reduced E2F activity by 49% and 73%, respectively. Quantitative RT-PCR analyses confirmed the downregulation of these DNA replication genes by 1,25(OH)2D3 in MC3T3-E1, GR, and VDRwt MEKs cells, but not in VDR-/- MEKs cells, showing that this 1,25(OH)2D3-driven antiproliferative action is of a general nature and depends on a functional VDR.

Identification of the human HEX1/ hExo1 gene promoter and characterization of elements responsible for promoter activity

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5′ to 3′ exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from −6240 to +1600 bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning −688 to +1600 bp that exhibits full transcriptional activity while a slightly shorter fragment from −658 to +1600 bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with −687 to −681 bp and −665 to −658 bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the −687 to −681 bp element, while transcription factors Sp1 and Sp3 recognize the −665 to −658 bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.

The RAD2 Domain of Human Exonuclease 1 Exhibits 5′ to 3′ Exonuclease and Flap Structure-specific Endonuclease Activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.