Flap Endonuclease Research Articles

Visualized Detection of Aldehyde Dehydrogenase 2 Gene Polymorphism By Serial Invasive Reaction Coupled with Gold Nanoparticle Probe Assembling

Conventional assays for identifying polymorphism of aldehyde dehydrogenase 2 (ALDH2) gene (rs671) are based on electrophoresis, fluorescence-labeled probes or sequencing, which are laborious and expensive. Herein, we proposed a visualized detection of ALDH2 genotype by PCR-based serial invasive reaction coupled with gold nanoparticle probe (GNP) assembling, in which target DNAs were firstly amplified by PCR, and then the alleles on the amplicons were identified by serial invasive reaction, and finally the genotyping results could be read out with naked eyes by observing the color changes caused by GNP assembling. If the allele-specific invasive probe matched the target sequence, the hairpin probe could be cleaved into two parts by Flap endonuclease 1 (FEN1). As a result, the GNPs could not be aggregated by the hairpin probe, keeping the solution red. Otherwise, the intact hairpin probe caused the aggregation of GNPs, leading to the color change from red to purple or clear. Therefore, the genotyping result could be read out with naked eyes by observing the color changes of the reaction. As low as 80 pg of genomic DNA could be accurately genotyped, and the genotyping results of 207 clinical samples were consistent with that of pyrosequencing, indicating that the proposed method has great potential in genotyping of personalized medicine-related single nucleotide polymorphism. A visualized method for detecting ALDH2 gene polymorphism site was established by coupling PCR-based serial invasive reaction coupled with gold nanoparticle probe (GNP) assembling. After reaction finished, the tube with target allele was red, and the tube without target allele was colorless.

Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation

More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5′ end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.

Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases.

Removal of ribonucleotides (rNMPs) incorporated into the genome by the ribonucleotide excision repair (RER) is essential to avoid genetic instability. In eukaryotes, the RNaseH2 is the only known enzyme able to incise 5′ of the rNMP, starting the RER process, which is subsequently carried out by replicative DNA polymerases (Pols) δ or ϵ, together with Flap endonuclease 1 (Fen-1) and DNA ligase 1. Here, we show that the DEAD-box RNA helicase DDX3X has RNaseH2-like activity and can support fully reconstituted in vitro RER reactions, not only with Pol δ but also with the repair Pols β and λ. Silencing of DDX3X causes accumulation of rNMPs in the cellular genome. These results support the existence of alternative RER pathways conferring high flexibility to human cells in responding to the threat posed by rNMPs incorporation.

Cancer Biomarker-Triggered Disintegrable DNA Nanogels for Intelligent Drug Delivery.

Even though various techniques have been developed thus far for targeted delivery of therapeutics, design and fabrication of cancer biomarker-triggered disintegrable nanogels, which are exclusively composed of nucleic acid macromolecules, are still challenging nowadays. Here, we describe for the first time our creation of intelligent DNA nanogels whose backbones are sorely disintegrable by flap endonuclease 1 (FEN1), an enzymatic biomarker that is highly overexpressed in most cancer cells but not in their normal counterparts. It is the catalytic actions of intracellular FEN1 on bifurcated DNA structures that lead to the cancer-specific disintegration of our DNA nanogels and controlled release of drugs in target cancer cells. Consequently, the brand-new strategies introduced in the current report could break new ground in designing drug carriers for eliminating unwanted side effects of chemotherapeutic agents and live-cell probes for cancer risk assessment, diagnosis, and prognosis.

Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response

Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven “undruggable”, Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with ∼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or “undruggable” targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of ∼2–8% and crystal structures from ∼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).

The oxidative stress responses caused by phthalate acid esters increases mRNA abundance of base excision repair (BER) genes in vivo and in vitro

The base excision repair (BER) pathway is an important defense response to oxidative DNA damage. It is known that exposures to phthalate esters (PAEs), including Dibutyl phthalate (DBP), Mono-(2-ethylhexyl) phthalate (MEHP), and Di-(2-ethylhexyl) phthalate (DEHP), cause reactive oxygen species-induced DNA damage and oxidative stress. Here, we determined the mRNA levels of BER pathway-related genes (ogg1, nthl1, apex1, parp1, xrcc1, lig3, ung, pcna, polb, pold, fen1, and lig1), pro-apoptotic gene (bax), and apoptotic suppressor gene (bcl2) in different PAEs-exposed zebrafish larvae and HEK293T cells. Further investigations were performed to examine reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD) activity, developmental toxicity, and cell viability after PAEs exposure in vivo and in vitro. The results showed that PAEs exposure can induce developmental abnormalities in zebrafish larvae, and inhibit cell viability in HEK293T cells. Additionally, we found that PAEs exposure results in the accumulation of ROS and the inhibition of SOD activation in vivo and in vitro. Notably, the mRNA levels of BER pathway-related genes (OGG1, NTHL1, APEX1, XRCC1, UNG, POLB, POLD, FEN1) were significantly upregulated after DBP or MEHP exposure, whereas the mRNA levels of NTHL1, UNG, POLB, POLD, and FEN1 were significantly altered in DEHP-treated HEK293T cells. In zebrafish, the mRNA levels of ogg1, pcna, fen1 and lig1 genes were increased after DBP or DEHP exposure, whereas the mRNA levels of nthl1, apex1, parp1, lig3, pcna and polb were decreased after MEHP exposure, respectively. Thus, our findings indicated that PAEs exposure can induce developmental toxicity, cytotoxicity, and oxidative stress, as well as activate BER pathway in vivo and in vitro, suggesting that BER pathway might play critical roles in PAEs-induced oxidative stress through repairing oxidative DNA damage.

Biological and clinical significance of flap endonuclease‑1 in triple‑negative breast cancer: Support of metastasis and a poor prognosis.

Flap endonuclease-1 (FEN1), a structure-specific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triple-negative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1-related transcripts in 198 patients with TNBC demonstrated that the polo-like kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.

DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes.

Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

The crystal structure of human XPG, the xeroderma pigmentosum group G endonuclease, provides insight into nucleotide excision DNA repair.

Nucleotide excision repair (NER) is an essential pathway to remove bulky lesions affecting one strand of DNA. Defects in components of this repair system are at the ground of genetic diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The XP complementation group G (XPG) endonuclease cleaves the damaged DNA strand on the 3′ side of the lesion coordinated with DNA re-synthesis. Here, we determined crystal structures of the XPG nuclease domain in the absence and presence of DNA. The overall fold exhibits similarities to other flap endonucleases but XPG harbors a dynamic helical arch that is uniquely oriented and defines a gateway. DNA binding through a helix-2-turn-helix motif, assisted by one flanking α-helix on each side, shows high plasticity, which is likely relevant for DNA scanning. A positively-charged canyon defined by the hydrophobic wedge and β-pin motifs provides an additional DNA-binding surface. Mutational analysis identifies helical arch residues that play critical roles in XPG function. A model for XPG participation in NER is proposed. Our structures and biochemical data represent a valuable tool to understand the atomic ground of XP and CS, and constitute a starting point for potential therapeutic applications.

PRMT1 is critical to FEN1 expression and drug resistance in lung cancer cells

The up-regulation of PRMT1 is critical to the cell growth and cancer progression of lung cancer cells. In our research, we found that PRMT1 is important to the DNA repair ability and drug resistance of lung cancer cells. To demonstrate the functions of PRMT1, we identified Flap endonuclease 1 (FEN1) as a post-translationally modified downstream target protein of PRMT1. As a major component of Base Excision Repair pathway, FEN1 plays an important role in DNA replication and DNA damage repair. However, the detailed mechanism of FEN1 up-regulation in lung cancer cells remains unclear. In our study, we identified PRMT1 as a key factor that maintains the high expression levels of FEN1, which is critical to the DNA repair ability and the chemotherapeutic drug resistance of lung cancer cells.

Succinylation at a key residue of FEN1 is involved in the DNA damage response to maintain genome stability.

Human flap endonuclease 1 (FEN1) is a structure-specific, multifunctional endonuclease essential for DNA replication and repair. Our previous study showed that in response to DNA damage, FEN1 interacts with the PCNA-like Rad9-Rad1-Hus1 complex instead of PCNA to engage in DNA repair activities, such as stalled DNA replication fork repair, and undergoes SUMOylation by SUMO-1. Here, we report that succinylation of FEN1 was stimulated in response to DNA replication fork-stalling agents, such as ultraviolet (UV) irradiation, hydroxyurea, camptothecin, and mitomycin C. K200 is a key succinylation site of FEN1 that is essential for gap endonuclease activity and could be suppressed by methylation and stimulated by phosphorylation to promote SUMO-1 modification. Succinylation at K200 of FEN1 promoted the interaction of FEN1 with the Rad9-Rad1-Hus1 complex to rescue stalled replication forks. Impairment of FEN1 succinylation led to the accumulation of DNA damage and heightened sensitivity to fork-stalling agents. Altogether, our findings suggest an important role of FEN1 succinylation in regulating its roles in DNA replication and repair, thus maintaining genome stability.

FEN1 endonuclease as a therapeutic target for human cancers with defects in homologous recombination

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.

Symmetrical dimethylation of H4R3: A bridge linking DNA damage and repair upon oxidative stress

The DNA lesions caused by oxidative damage are principally repaired by the base excision repair (BER) pathway. 8-oxoguanine DNA glycosylase 1 (OGG1) initiates BER through recognizing and cleaving the oxidatively damaged nucleobase 8-oxo-7,8-dihydroguanine (8-oxoG). How the BER machinery detects and accesses lesions within the context of chromatin is largely unknown. Here, we found that the symmetrical dimethylarginine of histone H4 (producing H4R3me2s) serves as a bridge between DNA damage and subsequent repair. Intracellular H4R3me2s was significantly increased after treatment with the DNA oxidant reagent H2O2, and this increase was regulated by OGG1, which could directly interact with the specific arginine methyltransferase, PRMT5. Arginine-methylated H4R3 could associate with flap endonuclease 1 (FEN1) and enhance its nuclease activity and BER efficiency. Furthermore, cells with a decreased level of H4R3me2s were more susceptible to DNA-damaging agents and accumulated more DNA damage lesions in their genome. Taken together, these results demonstrate that H4R3me2s can be recognized as a reader protein that senses DNA damage and a writer protein that promotes DNA repair.

The cooperative complex of Argonaute-2 and microRNA-146a regulates hepatitis B virus replication through flap endonuclease 1

AimHepatitis B virus (HBV) is a major cause of a variety of liver diseases. Existing antiviral drugs cannot eradicate HBV from our body, and the main reason is unclear on the molecular mechanism of HBV replication. Flap endonuclease 1 (FEN1) can repair relaxed circular DNA (HBV rcDNA) to covalently closed circular DNA (HBV cccDNA) that promotes HBV DNA replication, while its specific regulatory detail remains unclear. In addition, miR-146a is close related to regulation in HBV replication. This study aims to explore whether miR-146a regulates HBV cccDNA formation through FEN1. Main methodsWe investigated the expression of miR-146a, FEN1 and HBV copies in HBV stable replication cell line HepG2.2.15 and its parent cell line HepG2 transfected miR-146a and FEN1 plasmid by qRT-PCR and western blot, to identify the cooperation of Argonaute-2 (Ago2) and miR-146a by Ago2 siRNA and Ago2 RNA Binding Protein Immunoprecipitation (RIP). Key findingsCompared with the control group, we found that the expression of miR-146a was significantly up-regulated in HepG2.2.15, and the expression of FEN1 and HBV copies were also significantly up-regulated. On contrary, the expression of target gene of miR-146a, interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor-6 (TRAF6), was significantly decreased in HepG2.2.15. With the use of Ago2 siRNA and then Ago2 RIP, we found that Ago2 performed as a carrier for miR-146a to promote HBV replication. SignificanceThe results suggest a novel miR-146a → FEN1 → HBV DNA regulatory axis in HBV replication life. Ago2 cooperates with miR-146a to regulate the transcription and expression level of FEN1 protein through the downstream target gene IRAK1/TRAF6, and to promote HBV replication.

Flap endonuclease 1 (FEN1) as a novel diagnostic and prognostic biomarker for gastric cancer

Flap endonuclease 1 (FEN1) overexpression has been reported to be closely associated with cancer prognosis. However, its diagnostic and prognostic significance in gastric cancer (GC) has not yet been explored. FEN1 expression, its correlation with clinical parameters, and prognostic significance were investigated by data mining of The Cancer Genome Atlas (TCGA) datasets. Patients were divided into low- and high-expression groups using the median value of FEN1 expression as the cut-off. The diagnostic value of FEN1 expression in GC tissues was determined via receiver operating characteristic (ROC) curve analysis. Univariate and multivariate Cox regression analyses were used to identify the prognostic indicators. Gene set enrichment analysis (GSEA) was used to explore FEN1-related signalling pathways in GC. Furthermore, the Human Protein Atlas (HPA) database and GSE62254 dataset were used for further external validation. FEN1 was expressed at a higher level in GC tissues than in normal gastric tissues with high diagnostic accuracy (area under the ROC=0.909). Higher FEN1 expression was also validated at the protein level using the HPA database. High FEN1 expression in GC was correlated with older age (P<0.05). Patients with high FEN1 expression had a favourable prognosis compared to patients with low FEN1 expression (P=0.0048). Univariate and multivariate analyses revealed that FEN1 was an independent predictive factor associated with overall survival in both the TCGA cohort and the GSE62254 dataset (P=0.0004 and P=0.011, respectively). GSEA identified that the FEN1 expression was related to DNA replication, cell cycle, cytosolic and sensing pathways, oocyte meiosis, and the P53 signalling pathway. The results revealed high expression of FEN1 in GC; thus, it could be a promising early diagnostic and independent prognostic biomarker for GC.

Label-Free Imaging of Flap Endonuclease 1 in Living Cells by Assembling Original and Multifunctional Nanoprobe.

Flap endonuclease 1 (FEN1) becomes a potential tumor marker since it is closely related to cancer occurrence and development. Here, a poly dA20-mediated nanoprobe (AuNPs-poly dA20-poly dT20) was designed for FEN1 detection. Poly dA20 segment at the 3'- end of ssDNA adsorbed on AuNPs due to its strong affinity interaction with Au (stronger than Au-S bond), while the poly dT20 segment at the 5'- end overhangs. This nanoprobe not only worked as effective fluorescence quencher but also as the original nanosubstrate of FEN1. OliGreen adsorbed on poly dT20 emits strong green fluorescence because of its high sensitivity and selectivity toward thymine. However, it is quenched on the nanoprobe. In the presence of FEN1, it recognizes the overhanging poly dT20 segment and cleaves it efficiently, turning on the fluorescence of OliGreen. This indicates that the assembled nanoprobe is an effective artificial substrate to FEN1, although it is completely different from previously reported substrates that are all composed of dsDNA with a flap strand. This proposed nanoprobe was used to detect FEN1 not only in vitro but also in vivo. The method was simple, which avoided complex labeling procedures. It had a wide linear range from 0.05 U to 2 U, with the lowest detection limit of 0.007 U. Confocal imaging can distinguish cancer cells from normal cells, demonstrating its potential in clinical diagnostic and therapeutic monitoring.

Inhibition of FEN1 Increases Arsenic Trioxide-Induced ROS Accumulation and Cell Death: Novel Therapeutic Potential for Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, which is very difficult to treat and commonly develops resistance to chemotherapy. The following study investigated whether the inhibition of Flap Endonuclease 1 (FEN1) expression, the key enzyme in the base excision repair (BER) pathway, could improve the anti-tumor effect of arsenic trioxide (ATO), which is a reactive oxygen species (ROS) inducer. Our data showed that ATO could increase the expression of FEN1, and the knockdown of FEN1 could significantly enhance the sensitivity of TNBC cells to ATO both in vitro and in vivo. Further mechanism studies revealed that silencing FEN1 in combination with low doses of ATO might increase intracellular ROS and reduce glutathione (GSH) levels, by reducing the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); elevating ROS leaded to apoptosis and p38 and JNK pathway activating. In conclusion, our study suggested the combination of FEN1 knockdown and ATO could induce TNBC cell death by promoting ROS production. FEN1 knockdown can effectively decrease the application concentrations of ATO, thus providing a possibility for the treatment of TNBC with ATO.

A crystallization and preliminary X-ray diffraction study of the Arabidopsis thaliana proliferating cell nuclear antigen (PCNA2) alone and in a complex with a PIP-box peptide from Flap endonuclease 1.

DNA replication is an important event for all living organisms and the mechanism is essentially conserved from archaea, bacteria to eukaryotes. Proliferating cell nuclear antigen (PCNA) acts as the universal platform for many DNA transacting proteins. Flap endonuclease 1 (FEN1) is one such enzyme whose activity is largely affected by the interaction with PCNA. To elucidate the key interactions between plant PCNA and FEN1 and possible structural change of PCNA caused by binding of FEN1 at the atomic level, crystallization and preliminary studies of X-ray diffraction of crystals of Arabidopsis thaliana PCNA2 (AtPCNA2) alone and in a complex with a peptide derived from AtFEN1, which contains a typical PCNA-interacting protein (PIP)-box motif, were performed. Both peptide-free and peptide-bound AtPCNA2s were crystallized using the same reservoir solution but in different crystal systems, indicating that the peptide affected the intermolecular interactions in the crystals. Crystals of AtPCNA2 belonged to the hexagonal space group P63, while those of the peptide-bound AtPCNA2 belonged to the rhombohedral space group H3, both of which could contain the functional homo-trimers.

Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions

The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.