Apurinic endonuclease from Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Thielmann HW
PROVIDER: S-EPMC1162904 | biostudies-other | 1981 May
REPOSITORIES: biostudies-other
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Project description:MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLalpha (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLalpha, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLalpha is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSalpha, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLalpha endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLalpha ATPase activity or the ability of the protein to support assembly of the yMutLalpha.yMutSalpha.heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLalpha incision to the discontinuous strand of a nicked heteroduplex.
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Project description:SignificanceHuman apurinic/apyrimidinic endonuclease 1 (APE1, also known as REF-1) was isolated based on its ability to cleave at AP sites in DNA or activate the DNA binding activity of certain transcription factors. We review herein topics related to this multi-functional DNA repair and stress-response protein.Recent advancesAPE1 displays homology to Escherichia coli exonuclease III and is a member of the divalent metal-dependent α/β fold-containing phosphoesterase superfamily of enzymes. APE1 has acquired distinct active site and loop elements that dictate substrate selectivity, and a unique N-terminus which at minimum imparts nuclear targeting and interaction specificity. Additional activities ascribed to APE1 include 3'-5' exonuclease, 3'-repair diesterase, nucleotide incision repair, damaged or site-specific RNA cleavage, and multiple transcription regulatory roles.Critical issuesAPE1 is essential for mouse embryogenesis and contributes to cell viability in a genetic background-dependent manner. Haploinsufficient APE1(+/-) mice exhibit reduced survival, increased cancer formation, and cellular/tissue hyper-sensitivity to oxidative stress, supporting the notion that impaired APE1 function associates with disease susceptibility. Although abnormal APE1 expression/localization has been seen in cancer and neuropathologies, and impaired-function variants have been described, a causal link between an APE1 defect and human disease remains elusive.Future directionsOngoing efforts aim at delineating the biological role(s) of the different APE1 activities, as well as the regulatory mechanisms for its intra-cellular distribution and participation in diverse molecular pathways. The determination of whether APE1 defects contribute to human disease, particularly pathologies that involve oxidative stress, and whether APE1 small-molecule regulators have clinical utility, is central to future investigations.
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Project description:ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21-nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multispacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae.
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Project description:Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinson's disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkin's role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin-dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD-causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co-immunoprecipitation, proximity ligation assay, and co-localization in the cytoplasm. N-terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin-dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N-terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH-SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma. © 2016 Wiley Periodicals, Inc.
| S-EPMC5379662 | biostudies-literature
Project description:The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 ?M are required to inhibit APE1's endonuclease activity. To determine the role of E3330's negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1's redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC(50) values (8.5 and 7 ?M) lower than those produced with E3330 (20 and 55 ?M, respectively). Thus, E3330's negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.
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Project description:Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5' to an abasic site, giving a 3'-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and (13)C(beta)) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.
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Project description:Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential base excision repair enzyme that is upregulated in a number of cancers, contributes to resistance of tumors treated with DNA-alkylating or -oxidizing agents, and has recently been identified as an important therapeutic target. In this work, we identified hot spots for binding of small organic molecules experimentally in high resolution crystal structures of APE1 and computationally through the use of FTMAP analysis ( http://ftmap.bu.edu/ ). Guided by these hot spots, a library of drug-like macrocycles was docked and then screened for inhibition of APE1 endonuclease activity. In an iterative process, hot-spot-guided docking, characterization of inhibition of APE1 endonuclease, and cytotoxicity of cancer cells were used to design next generation macrocycles. To assess target selectivity in cells, selected macrocycles were analyzed for modulation of DNA damage. Taken together, our studies suggest that macrocycles represent a promising class of compounds for inhibition of APE1 in cancer cells.
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Project description:The repair of abasic sites that arise in DNA from hydrolytic depurination/depyrimidination of the nitrogenous bases from the sugar-phosphate backbone and the action of DNA glycosylases on deaminated, oxidized, and alkylated bases are critical to cell survival. Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1; aka APE1/ref-1) is responsible for the initial removal of abasic lesions as part of the base excision repair pathway. Deletion of APE-1 activity is embryonic lethal in animals and is lethal in cells. Potential inhibitors of the repair function of APE-1 were identified based upon molecular modeling of the crystal structure of the APE-1 protein. We describe the characterization of several unique nanomolar inhibitors using two complementary biochemical screens. The most active molecules all contain a 2-methyl-4-amino-6,7-dioxolo-quinoline structure that is predicted from the modeling to anchor the compounds in the endonuclease site of the protein. The mechanism of action of the selected compounds was probed by fluorescence and competition studies, which indicate, in a specific case, direct interaction between the inhibitor and the active site of the protein. It is demonstrated that the inhibitors induce time-dependent increases in the accumulation of abasic sites in cells at levels that correlate with their potency to inhibit APE-1 endonuclease excision. The inhibitor molecules also potentiate by 5-fold the toxicity of a DNA methylating agent that creates abasic sites. The molecules represent a new class of APE-1 inhibitors that can be used to probe the biology of this critical enzyme and to sensitize resistant tumor cells to the cytotoxicity of clinically used DNA damaging anticancer drugs.
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Project description:Human apurinic/apyrimidinic endonuclease (hApe1) encodes two important functional activities: an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFkappaB, HIF-1alpha, CREB, p53 and others. The BER function is highly conserved from prokaryotes (E. coli exonuclease III) to humans (hApe1). Here, we provide evidence supporting a redox function unique to mammalian Apes. An evolutionary analysis of Ape sequences reveals that, of the 7 Cys residues, Cys 93, 99, 208, 296, and 310 are conserved in both mammalian and non-mammalian vertebrate Apes, while Cys 65 is unique to mammalian Apes. In the zebrafish Ape (zApe), selected as the vertebrate sequence most distant from human, the residue equivalent to Cys 65 is Thr 58. The wild-type zApe enzyme was tested for redox activity in both in vitro EMSA and transactivation assays and found to be inactive, similar to C65A hApe1. Substitution of Thr 58 with Cys in zApe, however, resulted in a redox active enzyme, suggesting that a Cys residue in this position is indeed critical for redox function. In order to further probe differences between redox active and inactive enzymes, we have determined the crystal structures of vertebrate redox inactive enzymes, the C65A human Ape1 enzyme and the zApe enzyme at 1.9 and 2.3A, respectively. Our results provide new insights on the redox function and highlight a dramatic gain-of-function activity for Ape1 in mammals not found in non-mammalian vertebrates or lower organisms.
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Project description:MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX2EX4E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.
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