Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay.

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Homeostatic Milieu Induces Production of Deoxyribonuclease 1-like 3 from Myeloid Cells.

DNase 1-like 3 (DNase1L3), which belongs to DNase1 family, was originally identified as one of apoptosis- and necrosis-related endonucleases that fragmentate intranucleosomal DNA. A loss-of-function mutation has been reported in murine models of systemic lupus erythematosus (SLE) and in familial SLE patients. These reports suggest DNase1L3 plays an important role in the prevention of developing SLE; however, expression and function of DNase1L3 in human immune systems have been largely unclarified. As previous reports showed DNase1L3 is expressed in hematopoietic organs, we first analyzed expression levels of DNase1L3 in each subset of human peripheral blood cells by quantitative real-time PCR. Plasmacytoid dendritic cells showed the highest expression levels of DNase1L3 mRNA among peripheral blood cells. IL-4 enhanced DNase1L3 expression in monocytes, monocyte-derived dendritic cells, and monocyte-derived macrophages (MDMs), but not in T cells, B cells, or plasmacytoid dendritic cells. Together with IL-4, all-trans retinoic acid and apoptotic cells efficiently upregulated expression of DNalse1L3 in MDMs. As a result of intracellular signaling analysis, Jak1-IRS2-ERK/PI3K pathway was essential for IL-4-induced DNase1L3 expression. IL-4-treated monocyte-derived dendritic cells and MDMs secreted active DNase1L3 protein that could degrade liposome-DNA complexes, which were resistant to DNase1. Our results indicate DNase1L3 is secreted by innate immune cells and may play a critical role in the tissue homeostasis and on prevention of developing autoimmunity by degrading self-DNA.

Hookworms Evade Host Immunity by Secreting a Deoxyribonuclease to Degrade Neutrophil Extracellular Traps.

Hookworms cause a major neglected tropical disease, occurring after larvae penetrate the host skin. Neutrophils are phagocytes that kill large pathogens by releasing neutrophil extracellular traps (NETs), but whether they target hookworms during skin infection is unknown. Using a murine hookworm, Nippostrongylus brasiliensis, we observed neutrophils being rapidly recruited and deploying NETs around skin-penetrating larvae. Neutrophils depletion or NET inhibition altered larvae behavior and enhanced the number of adult worms following murine infection. Nevertheless, larvae were able to mitigate the effect of NETs by secreting a deoxyribonuclease (Nb-DNase II) to degrade the DNA backbone. Critically, neutrophils were able to kill larvae in vitro, which was enhanced by neutralizing Nb-DNase II. Homologs of Nb-DNase II are present in other nematodes, including the human hookworm, Necator americanus, which also evaded NETs in vitro. These findings highlight the importance of neutrophils in hookworm infection and a potential conserved mechanism of immune evasion.

Apoptotic Endonuclease EndoG Inhibits Telomerase Activity and Induces Malignant Transformation of Human CD4+ T Cells.

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.

Genetic and biochemical evidences reveal novel insights into the mechanism underlying Saccharomyces cerevisiae Sae2-mediated abrogation of DNA replication stress.

In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in double-strand break (DSB) repair, replication stress and telomere length maintenance. Another protein linked to DSB repair is Sae2, which regulates MRX persistence at DSBs. However, very little is known about its role in DNA replication stress and repair. Here, we reveal a crucial role for Sae2 in DNA replication stress. We show that different mutant alleles of SAE2 cause hypersensitivity to genotoxic agents, and when combined with Δmre11 or nuclease-defective mre11 mutant alleles, the double mutants are considerably more sensitive suggesting that the sae2 mutations synergize with mre11 mutations. Biochemical studies demonstrate that Sae2 exists as a dimer in solution, associates preferentially with single-stranded and branched DNA structures, exhibits structure-specific endonuclease activity and cleaves these substrates from the 5' end. Furthermore, we show that the nuclease activity is indeed intrinsic to Sae2. Interestingly, sae2G270D protein possesses DNA-binding activity, but lacks detectable nuclease activity. Altogether, our data suggest a direct role for Sae2 nuclease activity in processing of the DNA structures that arise during replication and DNA damage and provide insights into the mechanism underlying Mre11-Sae2-mediated abrogation of replication stressrelated defects in S. cerevisiae.

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling.

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [(3)H]myristic acid or [(3)H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.

SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr).

Evolutionarily conserved structure-selective endonuclease MUS81 forms a complex with EME1 and further associates with another endonuclease SLX4-SLX1 to form a four-subunit complex of MUS81-EME1-SLX4-SLX1, coordinating distinctive biochemical activities of both endonucleases in DNA repair. Viral protein R (Vpr), a highly conserved accessory protein in primate lentiviruses, was previously reported to bind SLX4 to mediate down-regulation of MUS81. However, the detailed mechanism underlying MUS81 down-regulation is unclear. Here, we report that HIV-1 Vpr down-regulates both MUS81 and its cofactor EME1 by hijacking the host CRL4-DCAF1 E3 ubiquitin ligase. Multiple Vpr variants, from HIV-1 and SIV, down-regulate both MUS81 and EME1. Furthermore, a C-terminally truncated Vpr mutant and point mutants R80A and Q65R, all of which lack G2 arrest activity, are able to down-regulate MUS81-EME1, suggesting that Vpr-induced G2 arrest is not correlated with MUS81-EME1 down-regulation. We also show that neither the interaction of MUS81-EME1 with Vpr nor their down-regulation is dependent on SLX4-SLX1. Together, these data provide new insight on a conserved function of Vpr in a host endonuclease down-regulation.

Promoting effect of licorice extract on spermatogonial proliferation and spermatocytes differentiation of neonatal mice in vitro.

Licorice (glycyrrhiza uralensis) is known as an herb with detoxication, and it has been widely used in clinical prescription of Oriental herbal medicine. Studies on the effects of licorice in the reproductive system were very rare, especially in spermatogenesis. In order to elucidate the effects of licorice on spermatogonial proliferation and spermatocyte differentiation during neonatal mice spermatogenesis, the organ culture model of testis tissue from neonatal C57BL/6N mice (born 6 d) was established. Then, in the presence of licorice extract (LE), the proliferation activity of spermatogonia was identified with the positive rate quantitative analysis of 5-bromo-2-deoxyuridine (BrdU) and anti-proliferating cell nuclear antigen (PCNA) antibody by immunohistochemical staining. The results showed that, compared to the control group, the percentage of positive cells by BrdU staining enhanced dramatically and that the expression of PCNA protein increased significantly in the spermatogonia from the LE group and showed a concentration-dependent manner (P < 0.05). This indicated that the LE can significantly promote the proliferation of spermatogonia in the spermatogenesis of neonatal mice. Furthermore, proteins related to spermatocyte differentiation, synaptonemal complex protein 3 (SCP3) and meiotic recombinant protein Spo11, were detected by immunohistochemical staining. The results showed that the differentiated spermatocyte in the LE group was significantly increased compared with that of the control group and showed a concentration-dependent manner (P < 0.05). The above results suggested that the LE can significantly accelerate the proliferation of spermatogonia and the differentiation of spermatocytes in the testicular tissue of the neonatal mice, which may be a potential drug for male infertility.

A novel Schistosoma japonicum endonuclease homologous to DNase II.

BACKGROUND: Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools. RESULTS: In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice. CONCLUSIONS: A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.

RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli.

For toxin/antitoxin (TA) systems, no toxin has been identified that functions by cleaving DNA. Here, we demonstrate that RalR and RalA of the cryptic prophage rac form a type I TA pair in which the antitoxin RNA is a trans-encoded small RNA with 16 nucleotides of complementarity to the toxin mRNA. We suggest the newly discovered antitoxin gene be named ralA for RalR antitoxin. Toxin RalR functions as a non-specific endonuclease that cleaves methylated and unmethylated DNA. The RNA chaperone Hfq is required for RalA antitoxin activity and appears to stabilize RalA. Also, RalR/RalA is beneficial to the Escherichia coli host for responding to the antibiotic fosfomycin. Hence, our results indicate that cryptic prophage genes can be functionally divergent from their active phage counterparts after integration into the host genome.

Nuclease-stimulated homologous recombination at the human ß-globin gene.

BACKGROUND: Mutations in the ß-globin gene (HBB) cause haemoglobinopathies where current treatments have serious limitations. Gene correction by homologous recombination (HR) is an attractive approach to gene therapy for such diseases and is stimulated by gene-specific endonucleases, including zinc finger nucleases (ZFNs). Customised nucleases targeting HBB have previously been shown to promote HR-mediated HBB modification in 0.3­60% of drug-selected cells, although frequencies among unselected cells, more relevant to the goal of correcting HBB in primary stem cells, have not been reported. METHODS: ZFNs targeting HBB were tested for HBB binding (two-hybrid assay) or HBB cleavage followed by inaccurate end joining (surveyor assay)in bacteria or human cancer cell lines, respectively. ZFN-stimulated HR was measured in cell lines by a modified fluorescence-based reporter assay or by targeted insertion of a drug-resistance marker into endogenous HBB confirmed by Southern analyses. RESULTS: Although the ZFNs that we assembled in-house showed limited potential, a commercially commissioned nuclease (ZFN4) enhanced HR mediated HBB modification in up to 95% of drug-selected cells. Among unselected cells, however, this frequency was less than 0.2%. Furthermore, ZFN4 cleaved HBB at an efficiency of 1­2% (surveyor assay) and enhanced the HR reporter assay 20-fold less efficiently than a control endonuclease. CONCLUSIONS: With ZFN4, we achieved higher efficiencies of HR-mediated HBB modification than previously reported for drug-selected cells. Our measurements of ZFN4-induced HR in unselected cells, however, suggest that improved nucleases must be developed if therapeutic HBB correction is to be achievable in primary stem cells.

HSF4 regulates DLAD expression and promotes lens de-nucleation.

HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2ß (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G.

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

Expression and purification of active mouse and human NEIL3 proteins.

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies.

[Expression, purification and characterization of non-specific Serratia nuclease in Escherichia coli].

To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 degrees C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 degrees C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11x10(6) U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.

ATM controls meiotic double-strand-break formation.

In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency.

UspB, a member of the sigma-S regulon, facilitates RuvC resolvase function.

A growing body of evidence shows that there is an intimate connection between proteins required for genome stability and stationary phase survival. In this work we show that the integral membrane protein UspB, a member of the RpoS regulon, is required for proper DNA repair as mutants lacking uspB are hypersensitive to several DNA damaging agents including ultraviolet light, mitomycin C, bleomycin and ciprofloxacin. Genetic and physical studies demonstrate that UspB acts in the RuvABC recombination repair pathway and removing uspB creates a phenocopy of the Holliday junction resolvase mutant, ruvC. Further, we show that the uspB mutant phenotype can be suppressed by ectopic overproduction of RuvC and that both ruvC and uspB mutants can be suppressed by inactivating recD. The fact that RuvABC-dependent repair requires UspB for proper activity suggests that the sigma-S regulon works together with DNA repair pathways under stress conditions to defend the cell against genotoxic stress.

The cdk5 kinase regulates the STAT3 transcription factor to prevent DNA damage upon topoisomerase I inhibition.

The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.

Production and characterization of recombinant protein preparations of Endonuclease G-homologs from yeast, C. elegans and humans.

Nuc1p, CPS-6, EndoG and EXOG are evolutionary conserved mitochondrial nucleases from yeast, Caenorhabditis elegans and humans, respectively. These enzymes play an important role in programmed cell death as well as mitochondrial DNA-repair and recombination. Whereas a significant interest has been given to the cell biology of these proteins, in particular their recruitment during caspase-independent apoptosis, determination of their biochemical properties has lagged behind. In part, biochemical as well as structural analysis of mitochondrial nucleases has been hampered by the fact that upon cloning and overexpression in Escherichia coli these enzymes can exert considerable toxicity and tend to aggregate and form inclusion bodies. We have, therefore, established a uniform E. coli expression system allowing us to obtain these four evolutionary related nucleases in active form from the soluble as well as insoluble fractions of E. coli cell lysates. Using preparations of recombinant Nuc1p, CPS-6, EndoG and EXOG we have compared biochemical properties and the substrate specificities of these related nucleases on selected substrates in parallel. Whereas Nuc1p and EXOG in addition to their endonuclease activity exert 5'-3'-exonuclease activity, CPS-6 and EndoG predominantly are endonucleases. These findings allow speculating that the mechanisms of action of these related nucleases in cell death as well as DNA-repair and recombination differ according to their enzyme activities and substrate specificities.