Multifunctional DNA Repair Enzyme Research Articles

A DNA/RNA hybrid fluorescent probe for high-throughput quantification of the activity of human apurinic/apyrimidinic endonuclease 1 in subcellular extracts

As a multifunctional DNA repair enzyme, human apurinic/apyrimidinic endonuclease 1 (APE1) plays a pivotal role in many cellular processes. In recent years, it has been recognized as a potential prognostic biomarker and a therapeutic target for many cancers and other diseases. In this work, we report a DNA/RNA hybrid-based fluorescent probe which allows rapid and high-throughput detection of APE1 in the nuclear, cytoplasmic, and mitochondrial extracts of different types of cultured cells without any purification steps. As a noncanonical substrate, the DNA/RNA hybrid probe possesses inherent specificity toward APE1 without the need of additional chemical modification. A substantial acceleration effect of the 3′-flanking mismatches enabled rapid digestion of the abasic sites in the DNA/RNA hybrid by APE1 at a comparable rate to those in natural double-stranded DNA. The hybrid probe showed adequately high sensitivity to the target enzyme (linear working range: 0.02–1.0 U/mL; detection limit: 0.01 U/mL) and superior resistance to interference from cellular proteins. The mechanism for the 3′-mismatch enhancement was also clarified via kinetic study and single-molecule fluorescence analysis. By using the probe, significantly varied subcellular distribution of APE1 in different cells were disclosed, showing the importance of quantitative measurement of APE1 activity in subcellular compartments for drug screening and evaluation of the treatment efficacy. The method was simple and robust, which offered an easily accessible molecular tool for high-throughput quantification of the subcellular APE1 activity. It may also facilitate the development of effective anticancer drugs.

A multifunctional DNA repair enzyme and magnetic dual-triggered theranostic nanosystem for intelligent drug delivery

Due to the high specificity and efficiency of enzymes, enzyme-responsive drug delivery systems are important in cancer therapy and diagnosis. Human apurinic/apyrimidinic endonuclease (APE1) is a representative DNA repair enzyme overexpressed in various cancer cells, which is also proven to be a promising stimuli in our previous studies. In this work, to realize targeted chemotherapy, an APE1 and magnetic dual-responsive nanocarrier was designed. The carrier is composed of Fe3O4@SiO2 nanoparticles and specially designed AP-DNA strand that can be effectively hydrolyzed by APE1. The chemotherapeutic drug doxorubicin (DOX) was loaded onto the AP-DNA to form the theranostic nanosystem (FS@DNA-DOX). As the AP-DNA was parted under the hydrolysis of APE1 in cancer cells, DOX was released to inhibit tumor growth. In vitro experiment results indicated that the nanosystem possesses a high specificity to APE1 and effectively release DOX in MCF-7 cells. In vivo experiments demonstrated that the targeted delivery of the nanosystem to the tumor site was successfully achieved and that strong tumor suppressor effects were observed. Overall, the FS@DNA-DOX is a multifunctional and theranostic nanosystem for the chemotherapy of cancer by increasing the therapeutic effect as well as reducing the toxicity of drugs.

The human AP-endonuclease 1 (APE1) is a DNA G-quadruplex structure binding protein and regulates KRAS expression in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients’ tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.

The APE1 redox inhibitor E3330 reduces collective cell migration of human breast cancer cells and decreases chemoinvasion and colony formation when combined with docetaxel.

The human apurinic/apyrimidinic endonuclease 1 (APE1) is an ubiquitous multifunctional DNA repair enzyme and a redox signalling protein. Our work addressed the inhibition of APE1 redox function using E3330, as single agent or in combination with docetaxel (DTX), in human breast cancer MDA-MB-231 cells. E3330 decreased the colony formation of DTX-treated cells. In addition, E3330 alone significantly reduced the collective cell migration as assessed by the wound-healing assay, whereas the combined treatment decreased chemoinvasion. These results suggest that the inhibition of APE1 redox function might have therapeutic potential by modulating cell migration and invasion in metastatic breast cancer.

The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

Abstract 342: The Antiapoptotic Function of APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 in Endothelial Cells Is Linked to Changes in Transcription Factor Activities and Independent of Its DNA Repair Domain

APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has both DNA repair and redox regulatory activity. Interestingly, APEX1 is also able to modulate transcription factor reduction, a prerequisite for DNA binding, independent of its intrinsic redox activity, probably by recruiting other reducing molecules like Thioredoxin-1 (TXN). APEX1 has been shown to exert anti-apoptotic properties in different cell lines and is localized in the cytosol and in the nucleus. So far it has not been studied whether APEX1 acts anti-apoptotic in primary human endothelial cells (EC) and whether this is dependent on its nuclear or cytosolic localization. After cloning human APEX1 and overexpression in EC we demonstrated that it inhibits not only basal but also H2O2-induced apoptosis. To further characterize the anti-apoptotic properties of APEX1 we generated mutants that lacked the C-terminal DNA repair domain (APEX1ΔC 1-127) or the N-terminal nuclear localization signal (NLS) (APEX1ΔN 21-318). We first analyzed the localization of the mutants by immunostaining. APEX1wt and APEX1ΔC 1-127 showed a predominant nuclear staining whereas APEX1ΔN 21-318 was mainly localized in the cytosol. With respect to the anti-apoptotic properties, only the mutant with intact NLS and redox domain APEX1ΔC 1-127 prevented apoptosis both under basal conditions as well as after H2O2 treatment similar to APEX1wt. The association between nuclear localization of APEX1wt and its anti-apoptotic properties in EC suggests that this function is linked to changes in transcription factor activities. It is known that the p65 subunit of the transcription factor complex NF-kB is activated by pro-apoptotic stimuli in EC. Using a specific DNA binding assay we could show that overexpression of APEX1wt and APEX1ΔC 1-127 reduced DNA binding of p65, whereas the NLS-deficient mutant had no effect. In conclusion, the anti-apoptotic activity of APEX1 in EC critically depends on its nuclear localization and redox-activity, is independent of its DNA repair domain and is at least partially due to suppression of NF-kB dependent, pro-apoptotic gene expression programs. In future studies we will analyze the impact of APEX1 on other transcription factors in concert with TXN.

APEX Nuclease (Multifunctional DNA Repair Enzyme) 1 Gene Asp148Glu Polymorphism and Cancer Risk: A Meta-Analysis Involving 58 Articles and 48903 Participants

BackgroundPolymorphisms in the APEX nuclease (multifunctional DNA repair enzyme) 1 gene (APEX1) may be involved in the carcinogenesis by affecting DNA repair. We aimed to summarize available data on the association of the APEX1 Asp148Glu (rs1130409) polymorphism with risk of multiple types of cancer via a meta-analysis.Methods and ResultsIn total, 58 qualified articles including 22,398 cancer patients and 26,505 controls were analyzed, and the data were extracted independently by two investigators. Analyses of the full data set indicated a marginally significant association of the APEX1 Asp148Glu polymorphism with cancer risk under allelic (odds ratio (OR)=1.05; 95% confidence interval (95% CI): 0.99-1.11; P=0.071), dominant (OR=1.09; 95% CI: 1.01-1.17; P=0.028), and heterozygous genotypic (OR=1.08; 95% CI: 1.01-1.16; P=0.026) models, with significant heterogeneity and publication bias. In subgroup analyses by cancer type, with a Bonferroni corrected alpha of 0.05/6, significant association was observed for gastric cancer under both dominant (OR=1.74; 95% CI: 1.2-2.51; P=0.003) and heterozygous genotypic (OR=1.66; 95% CI: 1.2-2.31; P=0.002) models. In subgroup analysis by ethnicity, risk estimates were augmented in Caucasians, especially under dominant (OR=1.11; 95% CI: 1.0-1.24; P=0.049) and heterozygous genotypic (OR=1.11; 95% CI: 0.99-1.24; P=0.063) models. By study design, there were no significant differences between population-based and hospital-based studies. In subgroup analysis by sample size, risk estimates were remarkably overestimated in small studies, and no significance was reached in large studies except under the heterozygous genotypic model (OR=1.23; 95% CI: 1.06-1.43; P=0.006, significant at a Bonferroni corrected alpha of 0.05/2). By quality score, the risk estimates, albeit nonsignificant, were higher in low-quality studies than in high-quality studies. Further meta-regression analyses failed to identify any contributory confounders for the associated risk estimates.ConclusionsOur findings suggest that APEX1 Asp148Glu polymorphism might be a genetic risk factor for the development of gastric cancer. Further investigations on large populations are warranted.

APEX nuclease multifunctional DNA repair enzyme 1 (APEX1)

APEX nuclease multifunctional DNA repair enzyme 1 (APEX1)

Habitual exercise increases resistance of lymphocytes to oxidant-induced DNA damage by upregulating expression of antioxidant and DNA repairing enzymes

The underlying mechanisms of adaptation from staying physically active are not completely revealed. This study examined the effects of 8 and 20 weeks of habitual voluntary exercise on the susceptibility of lymphocytes to oxidant-induced DNA damage, antioxidant enzyme activities in cardiac and skeletal muscles, and circulatory antioxidant profile. Forty young adult rats were randomly assigned to sedentary control and exercise groups for an experimental period of 8 or 20 weeks. Animals assigned to exercise groups were subjected to 24 h daily free access to an in-cage running wheel with circumference of 1.19 m. A magnetic digital counter was attached to the running wheel to record daily exercise distance run by the animals. Control rats were housed in cages without a running wheel, located next to the exercised animals. Body weight and food intake were recorded weekly. After the experimental periods of 8 and 20 weeks, blood, left ventricle, soleus and plantaris muscles were collected for analysis. No significant difference was found in plasma total antioxidant capacity between exercised and control animals in the 8 and 20 week groups according to our ferric reducing/antioxidant power (FRAP) analysis. However, modified FRAP for ascorbic acid (FRASC) analysis indicated that plasma ascorbic acid content was significantly increased by 46 and 34% in 8 and 20 week exercise groups, respectively, when compared with the corresponding control groups. Superoxide dismutase (SOD) activity was significantly elevated by 39% in erythrocytes of animals exercised for 8 weeks relative to control animals. In the 20 week exercise group, Glutathione peroxidase (GPx) activity in ventricle and plantaris was significantly upregulated by 477 and 290%, respectively, relative to control values. As demonstrated by comet assay, the oxidant-induced DNA damage was significantly reduced by 21 and 45% in lymphocytes of animals exercised for 8 and 20 weeks, respectively, when compared with the corresponding control lymphocytes. Our qRT-PCR analysis showed that the transcript expression of SOD2 was significantly elevated by 939% in lymphocytes of animals exercised for 8 weeks relative to control animals. Increased expressions of SOD2 (by 19%), catalase (25%), APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1; 46%), Protein kinase, DNA-activated, catalytic polypeptide (Prkdc; 9%) and O-6-methylguanine-DNA methyltransferase (Mgmt; 26%) were found in lymphocytes of animals exercised for 20 weeks relative to control rats. These results demonstrate that habitual exercise confers increased resistance of lymphocytes to oxidant-induced DNA damage, and this protective effect is possibly attributed to the regular exercise-induced elevated expression of antioxidant and DNA repairing enzymes.

Nuclear Redox Signaling

Reactive oxygen species have been described to modulate proteins within the cell, a process called redox regulation. However, the importance of compartment-specific redox regulation has been neglected for a long time. In the early 1980s and 1990s, many in vitro studies introduced the possibility that nuclear redox signaling exists. However, the functional relevance for that has been greatly disregarded. Recently, it has become evident that nuclear redox signaling is indeed one important signaling mechanism regulating a variety of cellular functions. Transcription factors, and even kinases and phosphatases, have been described to be redox regulated in the nucleus. This review describes several of these proteins in closer detail and explains their functions resulting from nuclear localization and redox regulation. Moreover, the redox state of the nucleus and several important nuclear redox regulators [Thioredoxin-1 (Trx-1), Glutaredoxins (Grxs), Peroxiredoxins (Prxs), and APEX nuclease (multifunctional DNA-repair enzyme) 1 (APEX1)] are introduced more precisely, and their necessity for regulation of transcription factors is emphasized.

Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

Comparison of methods for pharmacogenomics: SNaPshot, SNPstream UHT, Nanogen, and RFLP

The inter-individual variation of response to cancer chemotherapy and radiation therapy has a substantial genetic component and is a subject of pharmacogenetic studies. Our goal is to develop assays to analyze selected single nucleotide polymorphisms (SNPs) in genes with proven relevance for pharmacogenetics. Today's research in the field needs technological platforms for which the demands both in terms of quality and throughput are high. Pharmacogenomics utilizes various genotyping techniques as well as gene-expression studies in studying the effect of different pharmaceuticals as well as the pathways that influence them. Here we provide an overview of the platforms currently in use, and discuss their efficiency, precision and technological stability. The amount of hands-on time needed and the cost-effectiveness are discussed. We also compared the success rate of several methods. SNaPshot is a homogeneous-phase-based primer extension method and SNPstream UHT is a solid-phase-based primer extension method. The Nanogen chip employs binding of a biotinylated probe to a chip coated with hydrogel enriched with streptavidin. This universal design presents the possibility to perform both genotyping as well as gene-expression experiments, even on the same chip. Results were compared with the classical enzymatic restriction enzyme fragment polymorphism (RFLP) assay. The initial set included eight SNPs in the X-ray cross-complementing group 1 protein, XRCC1; APEX nuclease (multifunctional DNA repair enzyme) 1, APEX1; multidrug resistance gene, MDR1; gluthation-S-transferase P1, GSTP1; and tumor protein p53, TP53. Functional SNPs in these genes have been shown to influence the therapeutic effect of a series of anti-cancer drugs such as 5-fluorouracyl, doxorubicin, etoposide, thioguanine and irinotekan. Three series of 36 breast cancer patients treated with 5-fluorouracyl and mitomycin, of 92 breast cancer patients treated with doxorubicin and of 109 control individuals have been genotyped. This resulted in 1527 genotypes. Of these, 391 had been analysed by SNaPshot, 900 by SNPstream UHT, 454 by Nanogen and 966 by RFLP. The success rate was 91% for SNaPshot, 97% for SNPstream UHT, 96% for Nanogen, and 99% for RFLP. When comparing the methods pairwise, the lowest variation was observed between SNPstream UHT and Nanogen (0.4%). SNPstream UHT and Nanogen had similar rates of discrepancy when compared with RFLP: 3.2% and 2.6%, respectively. Close re-analysis of those samples revealed false calls due to incomplete cutting during the RFLP. The highest observed dis-concordance observed was between SNaPshot and SNPstream UHT (8.9%). Careful evaluation of genotyping quality is needed both for large epidemiological studies and smaller clinical trials, and especially in prospective pharmacogenetic studies of separate individuals in clinical settings.

Brain expression of apurinic/apyrimidinic endonuclease (APE/Ref-1) multifunctional DNA repair enzyme gene in the mouse with special reference to the suprachiasmatic nucleus

Multifunctional mammalian apurinic/apyrimidinic endonuclease (APE, also known as redox factor-1; Ref-1) repairs baseless sites of damaged DNA caused by oxidative stress and regulates the redox state of various DNA binding proteins. Here, we examined the expression of APE/Ref-1 m-RNA in the mouse brain by in situ hybridization. We detected APE/Ref-1 transcripts throughout the mouse brain particularly in the clock oscillating neurons of the suprachiasmatic nucleus (SCN), hippocampal pyramidal cells, granular cells, and in monoaminergic neurons. In the circadian center SCN, levels of APE/ref-1 mRNA transcripts were constantly high, and were not influenced by either circadian rhythms or by exposure to light.

Identification of the functional elements in the bidirectional promoter of the mouse O-sialoglycoprotein endopeptidase and APEX nuclease genes

The gene for mammalian O-sialoglycoprotein endopeptidase (Osgep) lies immediately adjacent to the gene for the APEX nuclease (Apex), a multifunctional DNA repair enzyme, in a head-to-head orientation. To clarify the regulation of these divergent genes, we studied their promoter regions with luciferase reporters. Deletion analysis of a fragment containing the entire mouse Apex gene suggested that cis-acting elements driving in the direction of Osgep are widely distributed in the mApex gene, in the antisense orientation. We investigated in detail cis-acting elements near the transcription initiation site of mOsgep. The spacer sequence between mOsgep and mApex was shown to have bidirectional promoter activity and it has been reported that two CCAAT boxes promote basal transcription in the direction of mApex. However, only one of the CCAAT boxes proximal to the transcription initiation site of mOsgep was important for transcription towards mOsgep. An Sp1-binding sequence was found to be involved in bidirectional transcription and a CRE/ATF-like sequence was shown to function as a repressor of mOsgep transcription. Quantitative RT-PCR showed that the mApex and mOsgep genes were expressed in all tissues examined and that expression of mOsgep was low compared with mApex.

Sequencing analysis of a putative human O-sialoglycoprotein endopeptidase gene (OSGEP) and analysis of a bidirectional promoter between the OSGEP and APEX genes

We performed cDNA and genomic cloning, sequencing and promoter analysis of the putative human O-sialoglycoprotein endopeptidase gene OSGEP (a homologue of gcp, a Pasteurella haemolytica A1 glycoprotease). The cloned OSGEP cDNA is 1311 nucleotides long, and encodes a protein consisting of 335 amino acids with predicted molecular mass of 36.4 kDa. The amino acid sequence of OSGEP showed 29.7% identity with that of P. haemolytica glycoprotease. The OSGEP gene is 7.75 kb long, consists of 11 exons and 10 introns, and lies immediately adjacent to the APEX gene (which encodes APEX nuclease, a multifunctional DNA repair enzyme) in 5′-to-5′ orientation. The promoter region of the OSGEP gene lacks the typical TATA box, but has putative regulatory elements in the CpG island. Northern blot analysis showed ubiquitous expression of the OSGEP gene in several tissues, and we observed similarities in expression patterns between OSGEP and APEX. In order to study the regulation of OSGEP gene expression, we analyzed the OSGEP promoter region by luciferase assay using HeLa cells. A functional region required for full transcription activity was narrowed down to a 23 bp region containing a CCAAT box. It has been reported that this CCAAT box promotes basal transcription in the APEX direction. We thus conclude that a bidirectional promoter containing a CCAAT box regulates transcription of both the OSGEP and APEX genes.

Functionally unrelated signalling proteins contain a fold similar to Mg 2+-dependent endonucleases

Here, I describe previously unidentified sequence and structural similarities between the family of Mg2+-dependent endonucleases and a divergent set of proteins that include phosphatases involved in cell-cycle regulation and signal transduction. The founding member of this endonuclease family is DNase I ( 1 Suck D. Oefner C. Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA. Nature. 1986; 321: 620-625 Crossref PubMed Scopus (285) Google Scholar ), joined by apurinic/apyrimidinic (AP) DNA-repair endonucleases that include exonuclease III (Exo III) and major human AP endonuclease (HAP1). Despite having <20% sequence identity and different cleavage preferences, these nucleases share the same catalytic residues and contain a similar four-layered α/β sandwich motif 2 Mol C.D. et al. Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature. 1995; 374: 381-386 Crossref PubMed Scopus (340) Google Scholar , 3 Gorman M.A. et al. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J. 1997; 16: 6548-6558 Crossref PubMed Scopus (288) Google Scholar . Recently, AP endonucleases were shown to have sequence similarity with the endonuclease domain of long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons 4 Feng Q. et al. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell. 1996; 87: 905-916 Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar and sphingomyelinases 5 Matsuo Y. et al. A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I. Protein Sci. 1996; 5: 2459-2467 Crossref PubMed Scopus (59) Google Scholar , indicating that this fold can be used for cleaving phosphoester bonds on different substrates.

Genomic structure of the rat major AP endonuclease gene (Apex) with an adjacent putative O-sialoglycoprotease gene (Prsmg1/Gcpl1) and a processed Apex pseudogene (Apexp1).

Genomic sequencing and chromosomal assignment of the gene encoding rat APEX nuclease, a multifunctional DNA repair enzyme, were performed. An active Apex gene and a processed pseudogene were isolated from a rat genomic library. The active Apex gene consists of 5 exons and 4 introns spanning 2.1 kb. The putative promoter region of the Apex gene lacks the typical TATA box, but contains CAAT boxes and a CpG island having putative binding sites for several transcription factors, such as Sp1, AP-2, GATA-1 and ATF. A putative O-sialoglycoprotease (a homologue of Pasteurella haemolytica glycoprotease, gcp; abbreviated as Prsmg1/Gcpl1) gene consisting of 11 exons and 10 introns spanning 7.3 kb lies immediately adjacent to the Apex gene in a 5'-to-5' orientation. The Apex gene locus was mapped to rat chromosome 15p12 using in situ hybridization. The processed pseudogene (designated as rat Apexp1) has a nucleotide sequence 87.1% identical to that of the rat Apex cDNA, although several stop codons interrupting the coding sequences and multiple nucleotide deletions were observed. The Apexp1 is located in an inactive LINE sequence. Calculation of nucleotide substitution rates suggests that the immediate, active progenitor of Apexp1 arose 23 million years ago and that the non-functionalization occurred 15 million years ago.

Characterization ofBacillus subtilisExoA Protein: a Multifunctional DNA-repair Enzyme Similar to theEscherichia coliExonuclease III

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.

Mutant AP endonuclease in patients with amyotrophic lateral sclerosis

Among the more frequent oxidative DNA injuries is the formation of abasic sites (AP sites) resulting from removal of purine or pyrimidine bases, estimated to occur at a rate of 1 x 10(4)/genome/24 h. A defect in DNA repair at this level could account for the accumulation of mutations and subsequent genome instability. We have identified missense mutations in the APE gene coding for a multifunctional DNA repair enzyme, AP endonuclease in eight of 11 patients with amyotrophic lateral sclerosis (ALS) and familial ALS. These mutations could affect the repair of abasic sites leading to the accumulation of mutations in neurons, resulting in their degeneration and death. Our findings implicate mutated AP endonuclease in the pathogenesis of ALS.

Focal nuclear hepatocyte response to oxidative damage following low dose thioacetamide intoxication.

Rats were treated with low doses of the hepatocarcinogen thioacetamide. Forty-eight hours following this treatment, microscopic foci of hepatic injury were observed, which were surrounded by a peripheral rim of histologically normal hepatocytes. These peripheral hepatocytes generally contained enlarged nuclei, and showed nuclear staining for 4-hydroxynonenal-protein adducts, indicative of nuclear oxidative damage. In these same hepatocytes, we also observed specific focal nuclear induction of mu-class glutathione-S-transferase and alcohol dehydrogenase I, two enzymes which are important in metabolism of 4-hydroxynonenal. Of particular interest was the concurrent nuclear induction of APE/ref-1, a multifunctional DNA repair enzyme which can function as a redox factor, and of the transcription factor Jun, whose DNA binding is facilitated by APE/ref-1. These results document an orchestrated focal nuclear response to oxidative damage produced by thioacetamide administration, and may relate to the permanent effects produced by this treatment.