ABSTRACT
Endonuclease V (EndoV) is a single-metal-dependent enzyme that repairs deaminated DNA nucleobases in cells by cleaving the phosphodiester bond, and this enzyme has proven to be a powerful tool in biotechnology and medicine. The catalytic mechanism used by EndoV must be understood to design new disease detection and therapeutic solutions and further exploit the enzyme in interdisciplinary applications. This study has used a mixed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) approach to compare eight distinct catalytic pathways and provides the first proposed mechanism for bacterial EndoV. The calculations demonstrate that mechanisms involving either direct or indirect metal coordination to the leaving group of the substrate previously proposed for other nucleases are unlikely for EndoV, regardless of the general base (histidine, aspartate, and substrate phosphate moiety). Instead, distinct catalytic pathways are characterized for EndoV that involve K139 stabilizing the leaving group, a metal-coordinated water stabilizing the transition structure, and either H214 or a substrate phosphate group activating the water nucleophile. In silico K139A and H214A mutational results support the newly proposed roles of these residues. Although this is a previously unseen combination of general base, general acid, and metal-binding architecture for a one-metal-dependent endonuclease, our proposed catalytic mechanisms are fully consistent with experimental kinetic, structural, and mutational data. In addition to substantiating a growing body of literature, suggesting that one metal is enough to catalyze P-O bond cleavage in nucleic acids, this new fundamental understanding of the catalytic function will promote the exploration of new and improved applications of EndoV.
Subject(s)
Nucleic Acids , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Lysine , Metals , Phosphates , WaterABSTRACT
Parkinson's disease (PD) is a common movement disorder caused by a characteristic loss of dopaminergic neurons in the substantia nigra and degeneration of dopamine terminals in the dorsal striatum. Previous studies have suggested that oxidative stress-induced DNA damage may be involved in PD pathogenesis, as steady-state levels of several types of oxidized nucleobases were shown to be elevated in PD brain tissues. These DNA lesions are normally removed from the genome by base excision repair, which is initiated by DNA glycosylase enzymes such as endonuclease VIII-like 1 (Neil1). In this study, we show that Neil1 plays an important role in limiting oxidative stress-induced degeneration of dopaminergic neurons. In particular, Neil1-deficient male mice exhibited enhanced sensitivity to nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and Neil1-deficient animals had higher levels of γH2AX-marked DNA damage than wild-type (WT) controls, regardless of treatment status. Moreover, MPTP-treated Neil1-/- male mice had slightly elevated expression of genes related to the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant pathway. Treatment with the Nrf2 activator, monomethyl fumarate, reduced PD-like behaviors and pathology in Neil1-/- male mice, suggesting that Neil1 is an important defense molecule in an oxidative cellular environment. Taken together, these results suggest that Neil1 DNA glycosylase may play an important role in limiting oxidative stress-mediated PD pathogenesis.
Subject(s)
DNA Glycosylases , Parkinson Disease , Male , Mice , Animals , Parkinson Disease/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Dopaminergic Neurons/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Substantia Nigra/pathology , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Mice, Inbred C57BL , Disease Models, Animal , Corpus Striatum/metabolismABSTRACT
Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.
Subject(s)
Deoxyribonuclease (Pyrimidine Dimer) , Sulfolobus , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Sulfolobus/genetics , Sulfolobus/metabolism , DNA Repair , DNA Damage , DNAABSTRACT
Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and removes bases, primarily oxidized pyrimidines, which have been damaged by endogenous oxidation or exogenous mutagenic agents. NEIL1 functions through a combined glycosylase/AP (apurinic/apyrimidinic)-lyase activity, whereby it cleaves the N-glycosylic bond between the DNA backbone and the damaged base via its glycosylase activity and hydrolysis of the DNA backbone through beta-delta elimination due to its AP-lyase activity. In our study we investigated our hypothesis proposing that the cancer resistance of the bowhead whale can be associated with a better DNA repair with NEIL1 being upregulated or more active. Here, we report the molecular cloning and characterization of three transcript variants of bowhead whale NEIL1 of which two were homologous to human transcripts. In addition, a novel NEIL1 transcript variant was found. A differential expression of NEIL mRNA was detected in bowhead eye, liver, kidney, and muscle. The A-to-I editing of NEIL1 mRNA was shown to be conserved in the bowhead and two adenosines in the 242Lys codon were subjected to editing. A mass spectroscopy analysis of liver and eye tissue failed to demonstrate the existence of a NEIL1 isoform originating from RNA editing. Recombinant bowhead and human NEIL1 were expressed in E. coli and assayed for enzymatic activity. Both bowhead and human recombinant NEIL1 catalyzed, with similar efficiency, the removal of a 5-hydroxyuracil lesion in a DNA bubble structure. Hence, these results do not support our hypothesis but do not refute the hypothesis either.
Subject(s)
Bowhead Whale , DNA Glycosylases , Escherichia coli Proteins , Lyases , Animals , Humans , Bowhead Whale/genetics , Bowhead Whale/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , DNA Repair , DNA Glycosylases/genetics , DNA Glycosylases/chemistry , DNA Glycosylases/metabolism , Cloning, Molecular , DNA , RNA, Messenger , Lyases/metabolism , Escherichia coli Proteins/genetics , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolismABSTRACT
Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA non-specifically and then slides along the DNA contour by one-dimensional diffusion. Opposite to the processive mechanism is distributive search, when an enzyme binds, samples and releases DNA without significant lateral movement. Many DNA glycosylases, the repair enzymes that excise damaged bases from DNA, use processive search to find their cognate lesions. Here, using a method based on correlated cleavage of multiply damaged oligonucleotide substrates we investigate the mechanism of lesion search by three structurally related DNA glycosylases-bacterial endonuclease VIII (Nei) and its mammalian homologs NEIL1 and NEIL2. Similarly to another homologous enzyme, bacterial formamidopyrimidine-DNA glycosylase, NEIL1 seems to use a processive mode to locate its targets. However, the processivity of Nei was notably lower, and NEIL2 exhibited almost fully distributive action on all types of substrates. Although one-dimensional diffusion is often regarded as a universal search mechanism, our results indicate that even proteins sharing a common fold may be quite different in the ways they locate their targets in DNA.
Subject(s)
DNA Glycosylases , Deoxyribonuclease (Pyrimidine Dimer) , Animals , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , DNA-Formamidopyrimidine Glycosylase/genetics , DNA-Formamidopyrimidine Glycosylase/metabolism , DNA Repair , DNA Glycosylases/genetics , DNA/metabolism , Oligonucleotides , Mammals/metabolismABSTRACT
Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.
Subject(s)
DNA Glycosylases , Deoxyribonuclease (Pyrimidine Dimer) , Cell Survival/genetics , DNA/metabolism , DNA Damage , DNA Glycosylases/metabolism , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Endonucleases/metabolism , Hydrogen Peroxide/pharmacology , Oxidative Stress , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Deoxyinosine (dI) is a highly mutagenic lesion that preferentially pairs with deoxycytidine during replication, which may induce A to G transition and ultimately contribute to carcinogenesis. Therefore, finding the site of dI modification in DNA is of great value for both basic research and clinical applications. Herein, we developed a novel method to sequence the dI modification site in DNA, which utilizes endonuclease V (EndoV)-dependent deamination repair to specifically label the modification site with biotin-14-dATP that allows the affinity enrichment of dI-bearing DNA for sequencing. We have achieved efficient determination of the location of the modified nucleotide in dI-bearing plasmid DNA with the assistance of EndoV-dependent deamination repair. We have also successfully applied this approach to locate the dI modification sites in the mitochondrial DNA of human cells. Our method should be generally applicable for genome-wide sequencing analysis of dI modifications in living organisms.
Subject(s)
DNA , Deoxyribonuclease (Pyrimidine Dimer) , DNA/genetics , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Humans , Inosine/analogs & derivativesABSTRACT
Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant's role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant.
Subject(s)
DNA Glycosylases , Carcinogenesis , DNA/metabolism , DNA Damage , DNA Glycosylases/metabolism , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli/genetics , Genomics , Humans , Purines , Pyrimidines/metabolism , Substrate SpecificityABSTRACT
Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.
Subject(s)
DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Fibroblasts/enzymology , Genomic Instability , Mutation, Missense , Animals , DNA/drug effects , DNA/metabolism , DNA Damage , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Fibroblasts/metabolism , Gene Knock-In Techniques , Mice , Mice, Mutant Strains , Mutagens/toxicity , Oxidative Stress , Vitamin K 3/toxicityABSTRACT
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.
Subject(s)
Catalytic Domain , DNA Repair , DNA/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Protein Domains , Amino Acid Sequence , Biocatalysis , DNA/genetics , DNA/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Humans , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Pyrimidines/metabolism , Sequence Homology, Amino AcidABSTRACT
Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.
Subject(s)
Arabidopsis/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Inosine/chemistry , RNA, Plant/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Binding Sites , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Gene Expression Regulation, Plant , RNA Editing , RNA, Plant/chemistry , Substrate SpecificityABSTRACT
POLE, POLD1, and NTHL1 are involved in DNA replication and have recently been recognized as hereditary cancer-predisposing genes, because their alterations are associated with colorectal cancer and other tumors. POLE/POLD1-associated syndrome shows an autosomal dominant inheritance, whereas NTHL1-associated syndrome follows an autosomal recessive pattern. Although the prevalence of germline monoallelic POLE/POLD1 and biallelic NTHL1 pathogenic variants is low, they determine different phenotypes with a broad tumor spectrum overlapping that of other hereditary conditions like Lynch Syndrome or Familial Adenomatous Polyposis. Endometrial and breast cancers, and probably ovarian and brain tumors are also associated with POLE/POLD1 alterations, while breast cancer and other unusual tumors are correlated with NTHL1 pathogenic variants. POLE-mutated colorectal and endometrial cancers are associated with better prognosis and may show favorable responses to immunotherapy. Since POLE/POLD1-mutated tumors show a high tumor mutational burden producing an increase in neoantigens, the identification of POLE/POLD1 alterations could help select patients suitable for immunotherapy treatment. In this review, we will investigate the role of POLE, POLD1, and NTHL1 genetic variants in cancer predisposition, discussing the potential future therapeutic applications and assessing the utility of performing a routine genetic testing for these genes, in order to implement prevention and surveillance strategies in mutation carriers.
Subject(s)
DNA Polymerase III/metabolism , DNA Polymerase II/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Neoplasms/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Female , Genetic Predisposition to Disease , Humans , MaleABSTRACT
DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.
Subject(s)
DNA Glycosylases/chemistry , DNA Glycosylases/metabolism , DNA Repair/physiology , Triage , Amino Acid Sequence , Binding Sites , Catalytic Domain , DNA/chemistry , DNA Damage , DNA Glycosylases/genetics , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Humans , Molecular Dynamics Simulation , Mutation , Protein Conformation , Substrate SpecificityABSTRACT
Efficient DNA repair is essential to maintain genomic integrity. An average of 30,000 base lesions per cell are removed daily by the DNA glycosylases of the base excision repair machinery. With the advent of whole genome sequencing, many germline mutations in these DNA glycosylases have been identified and associated with various diseases, including cancer. In this graphical review, we discuss the function of the NTHL1 DNA glycosylase and how genomic mutations and altered function of this protein contributes to cancer and aging. We highlight its role in a rare tumor syndrome, NTHL1-associated polyposis (NAP), and summarize various other polymorphisms in NTHL1 that can induce early hallmarks of cancer, including genomic instability and cellular transformation.
Subject(s)
Aging/metabolism , Colorectal Neoplasms/enzymology , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Aging/genetics , Colorectal Neoplasms/genetics , DNA/metabolism , DNA Glycosylases/metabolism , Genetic Predisposition to Disease , Germ-Line Mutation , Humans , Intestinal Polyposis/enzymology , Intestinal Polyposis/genetics , Polymorphism, GeneticABSTRACT
The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance, and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human endonuclease III (hNTH1). In the present study, we used Förster resonance energy transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid in vitro high-throughput screening for potential inhibitors of the hNTH1-YB1 complex. Two pilot screens were carried out, allowing the selection of several promising compounds exhibiting IC50 values in the low micromolar range. Interestingly, two of these compounds bind to YB1 and sensitize drug-resistant breast tumor cells to the chemotherapeutic agent, cisplatin. Taken together, these findings demonstrate that the hNTH1-YB1 interface is a druggable target for the development of new therapeutic strategies for the treatment of drug-resistant tumors. Moreover, beyond this study, the simple design of our biosensor defines an innovative and efficient strategy for the screening of inhibitors of therapeutically relevant protein-protein interfaces.
Subject(s)
Antineoplastic Agents/analysis , Biosensing Techniques/methods , Deoxyribonuclease (Pyrimidine Dimer)/antagonists & inhibitors , Protein Binding/drug effects , Y-Box-Binding Protein 1/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Drug Resistance, Neoplasm/drug effects , Fluorescence Resonance Energy Transfer/methods , Humans , MCF-7 Cells , Pilot Projects , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Y-Box-Binding Protein 1/metabolismABSTRACT
Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5' end. Treatment of the PCR products with endonuclease V generates 3' overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.
Subject(s)
DNA/genetics , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Gene Library , Inosine/analogs & derivatives , Oligonucleotides/genetics , Viral Proteins/genetics , Combinatorial Chemistry Techniques , DNA/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Inosine/chemistry , Inosine/genetics , Oligonucleotides/chemistry , Polymerase Chain Reaction , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm-1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli Proteins/metabolism , Iron-Sulfur Proteins/metabolism , Binding Sites/physiology , DNA Damage/physiology , DNA Glycosylases/metabolism , DNA Repair/physiology , Escherichia coli/metabolism , Oxidation-Reduction , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
Endonuclease V (EndoV) is a conserved inosine-specific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV-/- tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV-/- livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to fine-tune gene expression. The EndoV-/- tumor suppressive phenotype calls for related studies in human HCC.
Subject(s)
Deoxyribonuclease (Pyrimidine Dimer)/genetics , Liver Neoplasms, Experimental/genetics , Adenosine/metabolism , Animals , Antineoplastic Agents/pharmacology , Carcinogenesis , Cell Line , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Gene Expression , Humans , Inosine/metabolism , Liver/metabolism , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/pathology , Mice, Knockout , RNA Editing , RNA, Transfer/metabolism , Sequence Analysis, RNA , Sorafenib/pharmacologyABSTRACT
Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix-two-turn-helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6-30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5'-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination.
Subject(s)
DNA Glycosylases/chemistry , DNA Glycosylases/metabolism , DNA Repair/physiology , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA/chemistry , DNA/metabolism , DNA Damage , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-Formamidopyrimidine Glycosylase/metabolism , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , Humans , Nucleic Acid Conformation , Oxidative Stress , Transcription, Genetic/physiology , Uracil/analogs & derivatives , Uracil/metabolismABSTRACT
In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(ß-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.