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Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

ABSTRACT: Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg²⁺-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg²⁺ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

SUBMITTER: Kuznetsova AA

PROVIDER: S-EPMC8083922 | biostudies-literature |

REPOSITORIES: biostudies-literature

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AUF1 Recognizes 8-Oxo-Guanosine Embedded in DNA and Stimulates APE1 Endoribonuclease Activity.

Project description:Aims: The existence of modified ribonucleotide monophosphates embedded in genomic DNA, as a consequence of oxidative stress conditions, including 8-oxo-guanosine and ribose monophosphate abasic site (rAP), has been recently highlighted by several works and associated with oxidative stress conditions. Although human apurinic-apyrimidinic endodeoxyribonuclease 1 (APE1), a key enzyme of the base-excision repair pathway, repairs rAP sites and canonical deoxyribose monophosphate abasic sites with similar efficiency, its incision-repairing activity on 8-oxo-guanosine is very weak. The aims of this work were to: (i) identify proteins able to specifically bind 8-oxo-guanosine embedded in DNA and promote APE1 endoribonuclease activity on this lesion, and (ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. Results: By using an unbiased proteomic approach, we discovered that the AU-rich element RNA-binding protein 1 (AUF1) actively recognizes 8-oxo-guanosine and stimulates the APE1 enzymatic activity on this DNA lesion. By using orthogonal approaches, we found that: (i) the interaction between AUF1 and APE1 is modulated by H2O2-treatment; (ii) depletion of APE1 and AUF1 causes the accumulation of single- and double- strand breaks; and (iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. Innovation: These results establish unexpected functions of AUF1 in modulating genome stability and improve our knowledge of APE1 biology with respect to 8-oxo-guanosine embedded in DNA. Conclusion: By showing a novel function of AUF1, our findings shed new light on the process of genome stability in mammalian cells toward oxidative stress-related damages. Antioxid. Redox Signal. 39, 411-431.

| S-EPMC10517317 | biostudies-literature

Kinetic Features of 3'-5' Exonuclease Activity of Human AP-Endonuclease APE1.

Project description:Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.

| S-EPMC6225374 | biostudies-literature

AUF1 recognizes 8-oxo-guanosine embedded in DNA and stimulates APE1 endoribonuclease activity

Project description:The presence of rNMPs embedded in DNA represents a frequent genomic event able to destabilize the DNA backbone and to alter DNA replication and transcription. Usually, rNMPs are excised by RNase H2 and fully repaired by other enzymes belonging to the ribonucleotide excision repair pathway. The existence of modified rNMPs embedded in DNA, including 8-oxo-guanosine (8-oxo-G) and ribose monophosphate abasic site (rAP), have been recently highlighted by several works including ours. While eukaryotic RNase H2 is unable to process rAP and 8-oxo-G embedded in DNA, we demonstrated a role of APE1, a key enzyme of the base excision repair pathway, on these lesions. Notably, though APE1 activity on the repair of rAP sites results as efficient as the canonical dAP site, its incision repair activity on 8-oxo-G is very weak. Therefore, in order to guarantee an efficient repair activity of 8-oxo-G, additional processing mechanisms should be active in cells. In this work, by using an unbiased proteomic approach to isolate proteins able to recognize 8-oxo-G embedded in DNA, we discovered that AUF1 actively and specifically recognize 8-oxo-G and, interestingly, stimulates the APE1 enzymatic activity on this substrate. Although further structural analysis should be performed, it is reasonable that AUF1 facilitates the endoribonuclease activity of APE1 on 8-oxo-G by stabilizing the lesion substrate. Furthermore, by two orthogonal approaches including GST-pulldown and proximity ligation assay, we uncovered a novel protein-protein interaction between AUF1 and APE1. Remarkable, this interaction is modulated by H2O2-treatment, playing a putative biological role during cell response to oxidative stress.

2024-10-17 | PXD027252 | Pride

Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation.

Project description:The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.

| S-EPMC7094243 | biostudies-literature

Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII.

Project description:Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and β, δ-elimination of 3'- and 5'-phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5'-phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion "sensor", which is used by Endo VIII for recognition of a DNA damage site.

| S-EPMC5448014 | biostudies-literature

Endoribonuclease activity in hepatitis C virus-infected Huh7.5.1 cells

Project description:We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and endoribonuclease cleavage sites in host and viral RNAs during HCV infection of Huh7.5.1 cells

2022-08-06 | GSE72144 | GEO

Mutational Analysis

Project description:Mutational Analysis Revealed Polymorphic Variants in Exon 3 and Exon 4 of p53 Gene in Cervical Cancer

| PRJEB36236 | ENA

Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From <i>Thermobifida fusca</i> YX.

Project description:Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

| S-EPMC7835884 | biostudies-literature

Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

Project description:The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

| S-EPMC4552243 | biostudies-literature

Glioblastoma invasion and cooption depend on IRE1? endoribonuclease activity.

Project description:IRE1? is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. The protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes. In an orthotopic xenograft model of human glioma, invalidation of IRE1? RNase or/and kinase activities generated tumors with remarkably distinct phenotypes. Contrasting with the extensive angiogenesis observed in tumors derived from control cells, the double kinase/RNase invalidation reprogrammed mesenchymal differentiation of cancer cells and produced avascular and infiltrative glioblastomas with blood vessel co-option. In comparison, selective invalidation of IRE1? RNase did not compromise tumor angiogenesis but still elicited invasive features and vessel co-option. In vitro, IRE1? RNase deficient cells were also endowed with a higher ability to migrate. Constitutive activation of both enzymes led to wild-type-like lesions. The presence of IRE1?, but not its RNase activity, is therefore required for glioblastoma neovascularization, whereas invasion results only from RNase inhibition. In this model, two key mechanisms of tumor progression and cancer cell survival are functionally linked to IRE1?.

| S-EPMC4694804 | biostudies-other

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