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Trichinellosis is a foodborne zoonosis caused by Trichinella spiralis (T. spiralis) that not only causes considerable economic losses for the global pig breeding and food industries, but also seriously threats the health of human. Therefore, it is very necessary to develop an effective vaccine to prevent trichinellosis. In this study, the invasive Lactobacillus plantarum (L. plantarum) expressing fibronectin-binding protein A (FnBPA) was served as a live bacterial vector to deliver DNA to the host to produce a novel oral DNA vaccine. Co-expressing T. spiralis SS1 and murine interleukin-4 (mIL-4) of DNA vaccine were constructed and subsequently delivered to intestinal epithelial cells via invasive L. plantarum. At 10 days after the third immunization, the experimental mice were challenged with 350 T. spiralis infective larvae. The results found that the mice orally vaccinated with invasive L. plantarum harboring pValac-SS1/pSIP409-FnBPA not only stimulated the production of anti-SS1-specific IgG, Th1/Th2 cell cytokines, and secreted(s) IgA but also decreased worm burden and intestinal damage. However, the mice inoculated with invasive L. plantarum co-expressing SS1 and mIL-4 (pValac-SS1-IL-4/pSIP409-FnBPA) induced the highest protective immune response against T. spiralis infection. The DNA vaccine delivered by invasive L. plantarum provides a novel idea for the prevention of T. spiralis infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE., Competing Interests: Disclosures: P. Izmirly reported personal fees from GlaxoSmithKline outside the submitted work. J.P. Buyon reported personal fees from Bristol-Myers Squibb LN advisory board, personal fees from GSK panel on LN, personal fees from Amgen, and personal fees from Ventus outside the submitted work. B. Reizis reported being an advisor at Related Sciences. No other disclosures were reported., (© 2021 Hartl et al.)

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM., (Copyright © 2019 Costello, Cantillo and Kenter.)

Sepsis is one of the most significant challenges in intensive care units, which is associated with increased morbidity and mortality. Sepsis-associated encephalopathy (SAE) is a severe complication which can cause death and serious disabilities. Calcium signaling in astrocyte is essential for cellular activation and the potential resolution of infection or inflammation in SAE patients. The transient receptor potential melastatin 2 (TRPM2) channel has been identified as a unique fusion of a Ca 2+ -permeable nonselective cation channel, which plays an important role in inflammation and immune response. Because of its role as an oxidative stress sensor in astrocytes, we investigated the function of TRPM2 in inflammation mediators (interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α) release, Bcl-2/E1B-19 K-interacting protein 3 (BNIP3), apoptosis inducing factor (AIF) and Endonuclease G (Endo G) expression. We showed that TRPM2-KO mice, when intraperitoneally (i.p) injected with LPS, exhibited better neurologic assessment scores and decreased inflammatory injury in hippocampal neurons compared with wild-type (WT) mice. The absence of TRPM2 triggered less production of inflammatory mediators (IL-1β, IL-6, TNF-α) and decreased apoptosis related proteins (BNIP3, AIF, Endo G) expressions in response to LPS induced sepsis. Furthermore, TRPM2-deficient astrocytes (transfected with TRPM2 siRNA) upon LPS stimulation also induced decreased IL-1β, IL-6 and TNF-α level. Our data suggested that decreased production of inflammatory cytokines and apoptosis related proteins with TRPM2 deletion could regulate inflammatory stress and decrease inflammatory injury in hippocampal neurons, and consequently, ameliorate brain disorder., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Metastatic uveal melanoma is a deadly disease with no proven standard of care. Here we present a metastatic uveal melanoma patient with an exceptional high sensitivity to a PD-1 inhibitor associated with outlier CpG>TpG mutation burden, MBD4 germline deleterious mutation, and somatic MBD4 inactivation in the tumor. We identify additional tumors in The Cancer Genome Atlas (TCGA) cohorts with similar hypermutator profiles in patients carrying germline deleterious MBD4 mutations and somatic loss of heterozygosity. This MBD4-related hypermutator phenotype may explain unexpected responses to immune checkpoint inhibitors.

Conjugative plasmids are the main carriers of transmissible antibiotic resistance (AbR) genes. For that reason, strategies to control plasmid transmission have been proposed as potential solutions to prevent AbR dissemination. Natural mechanisms that bacteria employ as defense barriers against invading genomes, such as restriction-modification or CRISPR-Cas systems, could be exploited to control conjugation. Besides, conjugative plasmids themselves display mechanisms to minimize their associated burden or to compete with related or unrelated plasmids. Thus, FinOP systems, composed of FinO repressor protein and FinP antisense RNA, aid plasmids to regulate their own transfer; exclusion systems avoid conjugative transfer of related plasmids to the same recipient bacteria; and fertility inhibition systems block transmission of unrelated plasmids from the same donor cell. Artificial strategies have also been designed to control bacterial conjugation. For instance, intrabodies against R388 relaxase expressed in recipient cells inhibit plasmid R388 conjugative transfer; pIII protein of bacteriophage M13 inhibits plasmid F transmission by obstructing conjugative pili; and unsaturated fatty acids prevent transfer of clinically relevant plasmids in different hosts, promoting plasmid extinction in bacterial populations. Overall, a number of exogenous and endogenous factors have an effect on the sophisticated process of bacterial conjugation. This review puts them together in an effort to offer a wide picture and inform research to control plasmid transmission, focusing on Gram-negative bacteria.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.

CRISPR-Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR-Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1-2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1-2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1-2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Alterations in the apoptosis of immune cells have been associated with autoimmunity. Here, we have identified a homozygous missense mutation in the gene encoding the base excision repair enzyme Nei endonuclease VIII-like 3 (NEIL3) that abolished enzymatic activity in 3 siblings from a consanguineous family. The NEIL3 mutation was associated with fatal recurrent infections, severe autoimmunity, hypogammaglobulinemia, and impaired B cell function in these individuals. The same homozygous NEIL3 mutation was also identified in an asymptomatic individual who exhibited elevated levels of serum autoantibodies and defective peripheral B cell tolerance, but normal B cell function. Further analysis of the patients revealed an absence of LPS-responsive beige-like anchor (LRBA) protein expression, a known cause of immunodeficiency. We next examined the contribution of NEIL3 to the maintenance of self-tolerance in Neil3-/- mice. Although Neil3-/- mice displayed normal B cell function, they exhibited elevated serum levels of autoantibodies and developed nephritis following treatment with poly(I:C) to mimic microbial stimulation. In Neil3-/- mice, splenic T and B cells as well as germinal center B cells from Peyer's patches showed marked increases in apoptosis and cell death, indicating the potential release of self-antigens that favor autoimmunity. These findings demonstrate that deficiency in NEIL3 is associated with increased lymphocyte apoptosis, autoantibodies, and predisposition to autoimmunity.

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Detection of endogenous nucleic acids by cytosolic receptors, dependent on STING, and endosomal sensors, dependent on Unc93b1, can provoke inflammatory responses that contribute to a variety of autoimmune and autoinflammatory diseases. In DNase II-deficient mice, the excessive accrual of undegraded DNA leads to both a STING-dependent inflammatory arthritis and additional Unc93b1-dependent autoimmune manifestations, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. In this study, we use bone marrow chimeras to show that clinical and histological inflammation in the joint depends upon DNase II deficiency in both donor hematopoietic cells and host radioresistant cells. Additional features of autoimmunity in these mice, known to depend on Unc93b1 and therefore endosomal TLRs, also require DNase II deficiency in both donor and host compartments, but only require functional TLRs in the hematopoietic cells. Collectively, our data demonstrate a major role of both stromal and hematopoietic cells in all aspects of DNA-driven autoimmunity. These findings further point to the importance of cytosolic nucleic acid sensors in creating an inflammatory environment that facilitates the development of Unc93b1-dependent autoimmunity., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Arabidopsis thaliana CRT1 (compromised for recognition of Turnip Crinkle Virus) was previously shown to be required for effector-triggered immunity. Sequence analyses previously revealed that CRT1 contains the ATPase and S5 domains characteristic of Microchidia (MORC) proteins; these proteins are associated with DNA modification and repair. Here we show that CRT1 and its closest homologue, CRH1, are also required for pathogen-associated molecular pattern (PAMP)-triggered immunity, basal resistance, non-host resistance and systemic acquired resistance. Consistent with its role in PAMP-triggered immunity, CRT1 interacted with the PAMP recognition receptor FLS2. Subcellular fractionation and transmission electron microscopy detected a subpopulation of CRT1 in the nucleus, whose levels increased following PAMP treatment or infection with an avirulent pathogen. These results, combined with the demonstration that CRT1 binds DNA, exhibits endonuclease activity, and affects tolerance to the DNA-damaging agent mitomycin C, argue that this prototypic eukaryotic member of the MORC superfamily has important nuclear functions during immune response activation.

Relative DNase, RNase (efficiency of hydrolysis of ribo- and deoxyribooligonucleotides (ON)), and phosphatase (removal of the ON 5' terminal phosphate) catalytic activities of antibodies (AB) obtained after rabbit immunization by DNA, DNase I, and DNase II were compared. It is shown that electrophoretically homogeneous preparations of polyclonal AB from non-immunized rabbits did not exhibit such activities. Immunization of rabbits by DNA, DNase I, and DNase II results in generation of IgG abzymes that exhibit high activity in the ON hydrolysis reaction and even higher activity in cleavage of 5' terminal phosphate of ON. In this case K(m) values for supercoiled plasmid DNA and ON found in reactions of their AB-dependent nuclease hydrolysis and phosphatase cleavage of 5' terminal phosphate differ by 2-4 orders of magnitude. This shows that nuclease and phosphatase activities belong to different abzyme fractions within polyclonal AB. Thus, in this work data indicative of the possibility of a formation of antibodies exhibiting phosphatase activity after immunization of animals with DNA, DNase I, and DNase II, were obtained for the first time. Possible reasons for production of AB with phosphatase activity after immunization of rabbits with these immunogens are discussed.

New information has profoundly improved our insight into the processes that account for lupus nephritis. This review summarizes the data proving that secondary necrotic chromatin fragments are generated and retained in kidneys at time-points when the major renal nuclease Dnase-1 is selectively and severely downregulated. Second, we discuss data, which may indicate that nuclease deficiencies are not associated with autoimmunity to chromatin. Secondary to downregulation of renal Dnase-1, large chromatin fragment-immunoglobulin G complexes are accumulated in glomerular basement membranes of patients producing anti-chromatin autoantibodies. Exposure of chromatin in situ in glomeruli is the factor that renders anti-chromatin (anti-dsDNA and anti-nucleosome) antibodies nephritogenic. Without exposed chromatin, they circulate as non-pathogenic antibodies. This shows that acquired loss of renal Dnase-1 enzyme activity is a dominant event responsible for the progression of lupus nephritis into end-stage disease. Before the loss of Dnase-1, lupus-prone (NZB × NZW) F1 mice develop mild or silent nephritis with mesangial immune complex deposits, which correlates solely with onset of anti-dsDNA antibody production. The principal cellular and molecular requirements needed to produce these autoantibodies have been explained experimentally, but the mechanism(s) accounting for them in vivo in context of lupus nephritis have not yet been determined. However, published data show that defects in nucleases operational in apoptotic or necrotic cell death are not associated with the induction of nephritogenic anti-dsDNA autoantibodies. The data discussed in this study explain how an unusual exposure of chromatin may be a central factor in the evolution of lupus nephritis in (NZB x NZW) F1 mice, but not in promoting nephritogenic chromatin-specific autoimmunity.

The serum of patients with many autoimmune (AI) diseases contains small fractions of antibodies possessing both DNase and RNase activities. It was shown that immunization of rabbits with DNA, RNA, DNase I and RNase leads to production of antibodies with DNase and RNase activities. It is not known whether anti-idiotypic antibodies against DNase II can possess DNase or RNase activity. Electrophoretically and immunologically homogeneous polyclonal IgGs (pIgGs) from the sera of rabbits immunized with DNase II were obtained by sequential chromatography of the serum proteins on Protein A-Sepharose and gel filtration. It was shown for the first time that immunization of healthy rabbits with bovine DNase II produces IgGs with intrinsic DNase and RNase activities. IgGs from rabbits immunized with BSA or non-immunized animals were catalytically inactive. It was shown that approximately 10% of the total IgG DNase and RNase activities belong to anti-idiotypic antibodies to DNase II ( approximately 0.1% of total pIgGs), while 90% of the activities did not interact with Sepharose bearing antibodies against DNase II and might be antibodies to nucleic acids bound to DNase II. Affinity chromatography on DNA-cellulose using elution of antibodies with different concentration of NaCl and an acidic buffer separated catalytic IgGs into five antibody subfractions, three of which hydrolyzed RNA faster than DNA while two subfractions demonstrated only DNase activity. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain subfraction of antibodies against DNase II.

We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.

Bacterially expressed recombinant proteins are widely used for producing specific antibodies. Unfortunately, many recombinant proteins are recovered as insoluble materials, so-called inclusion bodies. Inclusion bodies are rather advantageous from a point of view of immunogens because fairly pure proteins can be feasibly extracted from the inclusion bodies. However, we encounter a problem with an insoluble protein when we make an antigen-immobilized column for affinity purification of antibodies because we need a soluble protein in usual immobilization methods. Histidine-tagged proteins can be bound to Ni(2+)-resins in buffer containing 6M guanidine-HCl, in which most insoluble proteins are solubilized. Taking advantage of this feature, we have successfully purified antigen-specific antibodies by directly using Ni(2+)-resins onto which denatured proteins are bound.

Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.

Two novel monoclonal antibodies (mAbs), hg302 and hg303, raised against a synthetic peptide corresponding to the basic domain of human DNase gamma, are characterized in detail. In Western blot analysis, hg303 recognizes both wild type and C-terminal Myc-His-tagged human DNase gamma, but does not cross-react with human DNase I family members, DNase I, DNase X, or DNAS1L2. On the other hand, dot blot analysis reveals the fine specificity of hg302; it recognizes human, mouse, and rat DNase gamma, but not other DNase I family DNases under non-denaturing conditions. Furthermore, hg302 efficiently immunoprecipitates wild type, but not C-terminal Myc-His-tagged, human DNase gamma from cell lysates. In immunohistochemical analysis, hg302 strongly recognizes DNase gamma in the nuclei of X-ray irradiation-induced apoptotic, but not normal rat thymocytes. The specific detection of DNase gamma in apoptotic nuclei is confirmed by indirect-immunofluorescence analysis using TNF-alpha-induced apoptotic HeLa S3 cells transfected with DNase gamma. These results, together with the observations that DNase gamma is present in normal thymocytes and its activity is unchanged during the apoptosis, suggest that some molecular change(s), which triggers the activation of DNase gamma, occurs in response to apoptotic stimuli in the basic domain, and hg302 specifically recognizes the activated DNase gamma in immunohistochemical analysis., (Copyright 2000 Academic Press.)

Schizosaccharomyces pombe ultraviolet damage endonuclease (UVDE or Uve1p) performs the initial step in an alternative excision repair pathway for UV-induced DNA damage. This DNA repair pathway was originally thought to be specific for UV damage. However, the broad substrate specificity of Uve1p suggests a more general role for this enzyme. Uve1p recognizes UV-induced bipyrimidine photoadducts and other non-UV-induced DNA adducts. Biochemical and genetic analysis also suggests that Uve1p may be involved in orchestrating mismatch repair in vivo. This study demonstrates that Uve1p recognizes and cleaves heteroduplex DNA with small unpaired loops but does not recognize loops six to eight nucleotides in length. In addition, the enzyme does not recognize DNA with palindromic insertions that could form base-paired hairpin structures. The cleavage efficiency of Uve1p depends on the distance of a mismatch from the DNA terminus, suggesting that the 3' terminus may contribute to the strand discrimination signal for Uve1p. These biochemical activities are discussed in the context of the role of Uve1p in DNA repair.

Holliday junction resolving enzymes bind specifically to four-way DNA junctions created by the process of homologous recombination, cleaving them to yield recombinant duplex DNA products. Homologous recombination is known to occur in the third domain of life, the archaea, and may constitute a simplified model for the corresponding eucaryal pathway, but has not been well characterised. Identification of a gene encoding an archaeal Holliday junction resolving enzyme, Hjc, has recently been reported in the euryarchaea, and an activity has been observed in the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Here we report the identification, heterologous expression and characterisation of the Hjc protein from Sulfolobus. We demonstrate that Sulfolobus has two distinct junction resolving enzymes, Hjc and Hje, with differing substrate specificities., (Copyright 2000 Academic Press.)

Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase-a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs., (Copyright 1998 Academic Press Limited.)

A polyclonal antibody against purified bull seminal plasma Ca2+, Mg2+-dependent endonuclease was raised in a rabbit. The antibody specifically cross-reacted with chromatin-bound Ca2+, Mg2+-dependent endonucleases from bovine thymus, human placenta, and bovine, rat and mouse liver in addition to the bovine seminal enzyme. The antibody did not cross-react with other endonucleases examined, including the acid-endonucleases from bovine thymus and liver, porcine spleen DNase II, micrococcal nuclease, and bovine pancreas DNase I, a known Ca2+ and Mg2+ requiring endonuclease. The present results indicate that this antibody specifically recognizes a class of so-called Ca2+, Mg2+-dependent endonuclease, which is localized in cell nuclei of various tissues and is probably involved in chromatin degradation during apoptosis. The antibody will be used to study the functional role of this class of endonuclease.

Abasic sites represent ubiquitous DNA lesions that arise spontaneously or are induced by DNA-damaging agents. They block DNA replication and are considered to be cytotoxic and mutagenic. The key enzymes involved in the repair of abasic sites are apurinic/apyrimidinic (AP) endonucleases which process these lesions in an error-free mechanism. To analyze the role of AP endonuclease in the protection of mammalian cells against DNA damaging agents, we have transfected both the human (APE) and the yeast (APN1) AP endonuclease in Chinese hamster cells and compared the effects of expression of these genes in stable transfectants as to survival of cells and formation of chromosomal aberrations. Although APE was markedly expressed on RNA and protein level, nuclear extracts of human APE transfectants did not show a higher AP endonuclease activity than the parental line and became not more resistant to the cell killing and clastogenic effect of methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). In contrast, cells transfected with the yeast APN1 gene expressed higher AP endonuclease activity and became clearly more resistant to the cytotoxic and chromosome breakage inducing activity of the agents. The results indicate that the excision repair capacity and correspondingly the mutagen resistance can be elevated by introducing, in mammalian cells, a yeast DNA repair gene and verify that AP sites are both cytotoxic and clastogenic lesions.

Cruciform-cutting endonuclease 1 (CCE1) is an X-solvase from yeast Saccharomyces cerevisiae [Kleff, S., Kemper, B. & Sternglanz, R. (1992) EMBO J. 11, 699-704]. We report here the purification of the cloned enzyme CCE1 to near homogeneity from over-expressing Escherichia coli cells. The purified protein has a globular shape and an apparent molecular mass of 38 kDa. CCE1 reacts specifically with branched DNAs, preferably with four-armed cruciforms. The enzyme linearizes native supercoiled DNA by cutting at the base of cruciform structures as they occur in derivatives of phage M13. Supercoiling was not required for cleavage per se and a relaxed circular DNA hybrid with a stable cruciform was linearized with the same relative cleavage efficiency. Fully synthetic cruciforms (four-armed X-junctions) were also good substrates for CCE1, provided a symmetric 6-bp sequence (in our case an EcoRI restriction site) was maintained at the junction. Consequently, a synthetic cruciform made from fully randomized oligonucleotide sequences was not a substrate for CCE1. In general, cleavage sites were found clustered in a characteristic pattern in each arm of a cruciform structure. A synthetic three-armed Y-junction was also cleaved by CCE1, but with a lower efficiency than the related four-armed construct. CCE1 resolves efficiently branched synthetic DNAs in vitro. The function is consistent with the idea that CCE1 is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA.

The Escherichia coli Uvr(A)BC endonuclease acts in a progression of several distinct steps accompanied by changes in the conformation of macromolecular constituents, the overall architecture of the complex, and its stoichiometry. In order to probe these structural changes, we generated monoclonal antibodies (mAbs) to Uvr proteins. The anti-UvrA mAb, A2D1, recognizing the N-terminal zinc-finger region of UvrA, and the anti-UvrB mAb, B2E2, having an epitope within the 43 C-terminal amino acids of UvrB, were purified and further characterized. It was found that A2D1 mAb interacts in solution both with UvrA-UvrB and UvrA-DNA complexes in the presence of the requisite ATP. This implies that the N-terminal zinc-finger of UvrA doesn't play a direct role in its interactions with UvrB and DNA. On the other hand, A2D1 does inhibit formation of the UvrB-damaged DNA preincision complex, apparently by preventing UvrB delivery by UvrA. The interaction of B2E2 with UvrA-UvrB and nucleoprotein complexes, including UvrB, suggests that the highly hydrophobic C-terminal domain of UvrB (i) doesn't participate in its interaction with UvrA, (ii) is accessible to this mAb in an intermediate UvrA-UvrB DNA helix-tracking complex, and (iii) seems to be directly involved in the formation of the preincision complex. These conclusions are supported by the finding that the neutralizing effect of A2D1 and B2E2 on the Uvr(A)BC endonuclease is significantly decreased if the preincision complex is preformed prior to mAbs addition.

Two enzyme forms of endonuclease (Sm 1 and Sm 2) strain B10M1 in 60 and 100 mg respectively have been isolated from the culture fluid Serratia marcescens. The chromatographic and electrophoretic properties and N-terminal amino acid residues are different for both enzymes. The purification procedure consists of dialysis and ion-exchange chromatography on DEAE- and phosphocellulose. The yield of nucleases Sm1 and Sm2 are 14% and 28% respectively. The antigenic differences of nucleases Sm1 and Sm2 have been found by cross immunoenzyme analysis.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.

In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10(4) clones) was first constructed in a new expression/secretion vector, using as inserts small fragments of HIV-1 DNA (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomyces cerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti-HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screening the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available.

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.

The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.

The inhibition of beef pancreatic deoxyribonuclease I activity with specific antibodies is studied using the hypochromic shift of DNA at 260 nm. The kinetics of this reaction reveals two reaction phases. The first phase, which ends within seconds, is reversible and follows second order kinetics. It leads quickly to the formation of big complexes. The second phase, which reaches completion within minutes before precipitation is detectable, is essentially irreversible and follows zero order kinetics. A model explaining these results is suggested.

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.

Eight hybridoma cell lines derived from fusion between myeloma X-63 and mouse splenocytes were found to secrete monoclonal antibodies against Ca/Mg-dependent endonuclease of human spleen cell nuclei. Two of them, termed N and S, were used in comparative research of enzymes from different organs and species of animals. The data obtained show that N and S antibodies recognize different antigenic determinants of the enzyme molecule. Cross-reactions of antibodies with different antigens having similar antigenic determinants, exist in Ca/Mg-endonuclease of such species as man, mouse, rat and cattle. The evolutionary conservatism of this enzyme is suggested. The data show that the existence of tissue-specific (thymus-specific and spleen-specific) isoforms of Ca/Mg-endonuclease of cell nuclei is possible.

Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.

Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.