Mammalian DNase Research Articles

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Intravenous Injection of Serratia marcescens Nuclease in Mice Induced Elevation of Synthetized and Secreted into the Serum Extracellular Ribonucleases

Deoxyribonucleases and ribonucleases are interest as potential anticancer compounds. Along with mammalian DNases and RNases the cancer suppressive effect was demonstrated for expressing the both activities nuclease from bacterium Serratia marcescens. The aim of the study was to examine how the appearing small amount of S.marcescens nuclease in blood can influence the level of serum DNase and RNase activities. Intravenous injection of S. marcescens nuclease in mice at the dose of 0.5µg /g of mouse was found to induce intensive rising RNA degrading activities in the serum. The induced activities differed from S.marcescens nuclease by the products of RNA degradation and requirement of magnesium cations. S.marcescens nuclease at the dose of 0.5µg /g of mouse did not both induce acute adverse effects and influenced on physical appearance and behavior in mice that evidenced a lack of the nuclease toxicity in vivo.

Secreted mammalian DNases protect against systemic bacterial infection by digesting biofilms.

Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.

In silico prediction of molecular mechanisms of toxicity mediated by the leptospiral PF07598 gene family-encoded virulence-modifying proteins.

Mechanisms of leptospirosis pathogenesis remain unclear despite the identification of a number of potential leptospiral virulence factors. We recently demonstrated potential mechanisms by which the virulence-modifying (VM) proteins-defined as containing a Domain of Unknown function (DUF1561), encoded by the PF07598 gene family-found only in group 1 pathogenic Leptospira-might mediate the clinical pathogenesis of leptospirosis. VM proteins belongs to classical AB toxin paradigm though have a unique AB domain architecture, unlike other AB toxins such as diphtheria toxin, pertussis toxin, shiga toxin, or ricin toxin which are typically encoded by two or more genes and self-assembled into a multi-domain holotoxin. Leptospiral VM proteins are secreted R-type lectin domain-containing exotoxins with discrete N-terminal ricin B-like domains involved in host cell surface binding, and a C-terminal DNase/toxin domain. Here we use the artificial intelligence-based AlphaFold algorithm and other computational tools to predict and elaborate on details of the VM protein structure-function relationship. Comparative AlphaFold and CD-spectroscopy defined the consistent secondary structure (Helix and ß-sheet) content, and the stability of the functional domains were further supported by molecular dynamics simulation. VM proteins comprises distinctive lectic family (QxW)3 motifs, the Mycoplasma CARDS toxin (D3 domain, aromatic patches), C-terminal similarity with mammalian DNase I. In-silico study proposed that Gln412, Gln523, His533, Thr59 are the high binding energy or ligand binding residues plausibly anticipates in the functional activities. Divalent cation (Mg+2-Gln412) and phosphate ion (PO4]-3-Arg615) interaction further supports the functional activities driven by C-terminal domain. Computation-driven structure-function studies of VM proteins will guide experimentation towards mechanistic understandings of leptospirosis pathogenesis, which underlie development of new therapeutic and preventive measures for this devastating disease.

Ehrlichia chaffeensis Uses an Invasin To Suppress Reactive Oxygen Species Generation by Macrophages via CD147-Dependent Inhibition of Vav1 To Block Rac1 Activation.

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.IMPORTANCEEhrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

Systemic lupus erythematosus: molecular cloning and analysis of recombinant DNase monoclonal κ light chain NGK-1.

Because DNase antibodies are cytotoxic, enter the nucleus and cause DNA fragmentation inducing cell death by apoptosis, they can play an important role in the pathogenesis of different autoimmune pathologies and especially systemic lupus erythematosus (SLE). The interesting goal of catalytic antibodies research is not only to study a possible biological role of such antibodies, but also to develop in future new human and animal therapies that use the advantages offered by abzymes. An immunoglobulin κ light chain library from SLE patients was cloned into a phagemid vector. Phage particles displaying recombinant monoclonal antibody light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA-cellulose. Sixteen of the 46 MLChs efficiently hydrolyzed DNA; one MLCh (approximately 27-28kDa) was expressed in Escherichia coli and purified by metal chelating and gel filtration. MLCh NGK-1 was electrophoretically homogeneous and demonstrated a positive answer with mouse IgGs against light chains of human antibodies after western blotting. SDS-PAGE in a gel containing DNA demonstrated that the MLCh hydrolyzes DNA and is not contaminated by canonical DNases. The DNase MLCh was activated by several metal ions. The protein sequence of the DNase MLCh has homology with mammalian DNases I and shares with them several identical or similar (with the same side chain functionality) important amino acid residues, which are necessary for DNA hydrolysis and binding of Mg(2+) and Ca(2+) ions. The affinity of DNA for this first example of a MLCh (K(M) = 0.3 microM) was 150- to 200-fold higher than for human DNase I.

The Structure of Human DNase I Bound to Magnesium and Phosphate Ions Points to a Catalytic Mechanism Common to Members of the DNase I-like Superfamily

Recombinant human DNase I (Pulmozyme, dornase alfa) is used for the treatment of cystic fibrosis where it improves lung function and reduces the number of exacerbations. The physiological mechanism of action is thought to involve the reduction of the viscoelasticity of cystic fibrosis sputum by hydrolyzing high concentrations of DNA into low-molecular mass fragments. Here we describe the 1.95 Å resolution crystal structure of recombinant human DNase I (rhDNase I) in complex with magnesium and phosphate ions, both bound in the active site. Complementary mutagenesis data of rhDNase I coupled to a comprehensive structural analysis of the DNase I-like superfamily argue for the key catalytic role of Asn7, which is invariant among mammalian DNase I enzymes and members of this superfamily, through stabilization of the magnesium ion coordination sphere. Overall, our combined structural and mutagenesis data suggest the occurrence of a magnesium-assisted pentavalent phosphate transition state in human DNase I during catalysis, where Asp168 may play a key role as a general catalytic base.

Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages

Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.

Two N-Linked Glycosylation Sites (Asn18 and Asn106) Are Both Required for Full Enzymatic Activity, Thermal Stability, and Resistance to Proteolysis in Mammalian Deoxyribonuclease I

Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

Cloning and structural analysis of the full-length cytolethal distending toxin (cdt) gene operon from Campylobacter lari

Polymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdt gene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lari isolates using a new PCR primer pair constructed in silico. Three closely spaced and putative ORFs for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic ρ-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and cdtB and a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtA and cdtB and the non-coding region of six base pairs occurring between cdtB and cdtC. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six in cdtB and two in cdtC among the seven isolates (including C. lari RM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtB and mammalian DNase I were completely conserved in the cdtB gene locus in the 26 C. lari isolates, as well as in C. jejuni and C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC; n=10) using the primer pair.

DNase X Is a Glycosylphosphatidylinositol-anchored Membrane Enzyme That Provides a Barrier to Endocytosis-mediated Transfer of a Foreign Gene

DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, beta-galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: Biochemical and molecular analysis of equine DNase I

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types — pancreatic, parotid and pancreatic–parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic–parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.

Cloning and expression of deoxyribonuclease II from chicken

Acid endonucleases of the deoxyribonuclease II (DNase II, EC 3.1.22.1) family have been implicated in the degradation of DNA from apoptotic cell corpses formed in the process of normal mammalian development. Although a predicted DNase II has been detected in the chicken through expressed sequence tag (EST) analysis, to date no homolog of these important enzymes has been identified in vivo in any avian species. Here we report the cloning and expression of DNase II from the chicken, Gallus gallus. When expressed, the 363 amino acid glycoprotein is observed to be approximately 45 kDa in size and to exhibit DNA hydrolytic activity at pH 5 consistent with DNase II in other species. Furthermore, chicken DNase II sequence is compared with an identified partial sequence from the zebra finch, Taeniopygia guttata, as well as the previously identified homologs found in the fowlpox and canarypox viruses and the previously cloned mammalian DNases II. Through analysis of its amino acid sequence, comparative gene structure, and conserved synteny, chicken DNase II appears to represent a member of the DNase IIβ subfamily and the apparent lack of a DNase IIα homolog in the chicken has important evolutionary implications for the study of this gene family.

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Molecular, biochemical and immunological analyses of canine pancreatic DNase I.

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.

Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus

The anthelmintic fenbendazole (FBZ) induces nuclear DNA fragmentation (DF) in intestinal cells of Haemonchus contortus. The DNA fragments had 3 ′-OH, which suggests involvement of a neutral DNase. To identify candidate DNase(s) involved, DNase activity in H. contortus intestine and other worm fractions was characterized relative to classic DNases I (neutral) and II (acidic). Seven distinct DNase activities were identified and had M rs of 34, 36, 37 or 38.5 kDa on zymographic analysis. The different activities were distinguished according to pH requirement, sensitivity to 10 mM EDTA and worm compartment. Activities of intestinal DNases at 34, 36 and 38.5 kDa were sensitive to EDTA at pH 5.0 and 7.0. Sensitivity to EDTA at pH 5.0 was unexpected compared to classic acidic DNase II activity, suggesting unusual properties of these DNases. In whole worms, however, the activities at 36 and 38.5 kDa were relatively insensitive to EDTA, indicating predominance of DNases that are distinct from the intestine. The activity at 37 kDa in excretory/secretory products had an acidic pH requirement and was insensitive to EDTA, resembling classic acidic DNase activity. Under conditions of pH 5.0 and 7.0, intestinal DNases produced 3 ′-ends that could be labeled by terminal deoxynucleotidyl transferase, indicating presence of 3 ′-OH. The labeling of 3 ′-ends at pH 5.0, again, was unexpected for acidic DNase activity. These results and several other activities suggest that multiple H. contortus DNases have characteristics distinct from the classic mammalian DNases I and II. Treatment of H. contortus with FBZ did not induce any detectable DNase activities distinct from normal intestine, although relative activities of intestinal DNases appear to have been altered by this treatment. Abbreviation: DNase, deoxyribonuclease; DF, DNA fragmentation; BZ, benzimidazole anthelmintics; TdT, terminal deoxynucleotidyl transferase; ATA, aurintricarboxylic acid

Chicken deoxyribonuclease: purification, characterization, gene cloning and gene expression.

Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn2+ and Mg2+ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu2+, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca2+ at pH 8, chicken DNase resisted to the trypsin or beta-mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, approximately 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5'-untranslated region and 166 of the 3' and, in the 5'-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 +/- 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.

A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability.

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild-type snake DNases I with Leu130 were more thermally unstable than wild-type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild-type counterparts and similar to mammalian wild-type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild-type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+-binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473-480]. Thus, it is plausible that the thermally stable wild-type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold-blooded to warm-blooded vertebrates.

Drosophila acid DNase is a homolog of mammalian DNase II

Mammalian DNase II enzymes and the Caenorhabditis elegans homolog NUC-1 have recently been shown to be critically important during engulfment-mediated clearance of DNA. In this report, we describe the cloning and characterization of the gene encoding Drosophila DNase II. Database queries using the C. elegans NUC-1 protein sequence identified a highly homologous open reading frame in Drosophila (CG7780) that could encode a similar enzyme. Analysis of crude protein extracts revealed that wild-type Drosophila contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC1, while the same activity was markedly reduced in an acid DNase hypomorphic mutant line. Furthermore, the pattern of cleavage products generated from an end-labeled substrate by hypomorphic-line extracts was significantly altered in comparison to the pattern generated by wild-type extracts. Sequence analysis of CG7780 DNA and mRNA revealed that the hypomorphic line contains a missense mutation within the coding region of this gene. Additionally, Northern analysis demonstrated that CG7780 expression is normal in the mutant line, which in combination with the lowered/altered enzymatic activity and sequencing data suggested a defect in the CG7780 protein. To conclusively determine if CG7780 encoded the Drosophila equivalent of DNase II/NUC-1, transgenic lines expressing wild-type CG7780 in the mutant background were generated and subsequently shown to complement the mutant phenotype. Our results, therefore, provide compelling evidence that the predicted gene CG7780 encodes Drosophila DNase II (dDNase II), an enzyme related in sequence and activity to mammalian DNase II. Interestingly, overexpression of CG7780 both ubiquitously and in specific tissues failed to elicit any discernable phenotype.

Resistance of 3'-phosphoglycolate DNA ends to digestion by mammalian DNase III.

An essential step in the repair of free radical-mediated DNA strand breaks is the removal of sugar fragments such as phosphoglycolate from the 3' termini. While the abasic endonuclease Ape1 can remove phosphoglycolate from single-strand breaks in double-stranded DNA, an enzyme capable of removing it from 3' overhangs of double-strand breaks has yet to be identified. We therefore tested DNase III, the predominant 3' --> 5' exonuclease in mammalian cell extracts, for possible 3'-phosphoglycolate-removing activity. However, all 3'-phosphoglycolate substrates, as well as a 3'-phosphate substrate, were resistant to DNase III under conditions in which the analogous 3'-hydroxyl substrates were extensively degraded. The DNA end-binding protein Ku (an equimolar mixture of Ku70, now known as G22P1, and Ku86, now known as XRCC5) did not alter the resistance of the 3'-phosphoglycolate substrates, but the protein modulated the susceptibility of 3'-hydroxyl substrates, allowing DNase III to remove a 3' overhang but inhibiting digestion of the double-stranded portion of the substrate.