Biochemical characterization of <i>Anabaena</i> sp. strain PCC 7120 non‐specific nuclease NucA and its inhibitor NuiA

Meiß, Gregor; Franke, Ingo; Gimadutdinow, Oleg; Urbanke, Claus; Pingoud, Alfred

doi:10.1046/j.1432-1327.1998.2510924.x

Cited by 50 publications

(59 citation statements)

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“…Nonspecific nuclease family members cleave all types of nucleic acids with similar specificity for RNA and for both ss and dsDNA substrates (Friedhoff et al, 1996;Ho and Liao, 1999;Meiss et al, 1998). However, some nucleases showing little sequence specificity do recognize certain structural features of their respective DNA substrates other than ss/ds DNA.…”

Section: Introductionmentioning

confidence: 99%

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

Calvo

Ribeiro

2006

Journal of Experimental Biology

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SUMMARY Previous analysis of the salivary gland transcriptome of Culex quinquefasciatus showed the potential presence of an endonuclease with sequence similarities to shrimp, crab and two tsetse salivary proteins. Indeed, not only was the cloned cDNA shown to encode an active double-stranded endonuclease, but also the same activity was demonstrated to be secreted by salivary glands of Cx. quinquefasciatus. Preliminary studies with salivary gland extracts confirmed the presence of a highly active nuclease. This enzyme was shown to be present in the saliva of female mosquitoes by allowing starved mosquitoes to probe DNA-containing agarose gel. The recombinant Cx. quinquefasciatus endonuclease (CuquEndo) produced in mammalian cells showed no sequence specificity for DNA substrate except that it only cleaves double-stranded DNA. Recombinant Cx. quinquefasciatusendonuclease was active in the presence of Mg2+ ions at pH 7.0-8.0,but no endonuclease activity was detected in the presence of calcium ions. The final hydrolysis products of this enzyme, detected by ion exchange chromatography, yielded DNA fragments ranging form 8-12 base pairs. Although endonucleases have been associated with a variety of cellular functions, their role in mosquito saliva is not clear. This female-specific secreted endonuclease may assist blood meal intake by lowering the local viscosity created by the release of host DNA in the bite site and/or acting as an indirect anticoagulant factor by producing a defibrotide-like mixture of DNA haptamers.

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Section: Introductionmentioning

confidence: 99%

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

Calvo

Ribeiro

2006

Journal of Experimental Biology

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“…PCC 7120 (1) is a member of a family of highly active, divalent metal ion-dependent, nonspecific nucleases, which are characterized by the DRGH prosite motif. NucA is able to degrade both single-and double-stranded DNA and RNA and functions optimally in the presence of Mn 2ϩ or Mg 2ϩ ions (2). The extracellular nuclease from Serratia marcescens (3), yeast Nuc1 (4), mitochondrial EndoG (5), and Streptococcus pneumoniae EndA (6) are some of the other family members.…”

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confidence: 99%

“…Instead, NucA is paired with a specific inhibitor, NuiA, analogous to the colicin immunity proteins in E. coli (17). NuiA protects the cell from nuclease action by forming a very stable NucA⅐NuiA complex (2,18,19). The three-dimensional structure of NuiA has recently been determined (20).…”

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Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Ghosh

Meiß

Pingoud

et al. 2005

Journal of Biological Chemistry

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Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single-and doublestranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the ⌬(2-24),D121A mutant of NucA in the presence of Zn 2؉and Mn 2؉ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a ␤␤␣ metal finger motif and a hydrated Mn 2؉ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites ϳ26 Å apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg 57 in Serratia, the structure determined here indicates that Arg 93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the ␤␤␣ metal finger nuclease family and available mutational data on NucA, we propose that His 124 acts as a catalytic base, and Arg 93 participates in the catalysis possibly through stabilization of the transition state.

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“…2, DNA supplemented with Cu# + and Zn# + [either without (lane 1) or with (lane 2) added nuclease] also appeared differently after electrophoresis and staining, perhaps suggesting a structural change in the DNA. Obviously, caution is needed when assessing the ability of a metal to act as a co-factor Values for kinetic parameters of other enzymes are from Friedhoff et al (1996) and Meiss et al (1998). with any nuclease assay.…”

Section: Cell Fractionation and Nuclease Localizationmentioning

confidence: 99%

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo

MacLellan

Forsberg

2001

Microbiology

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DNase A is a non-specific endonuclease of Fibrobacter succinogenes. The enzyme was purified to homogeneity and its properties studied both in vitro and in vivo. Magnesium but not calcium was essential for nucleolytic activity. Manganese ions substituted for magnesium but were less stimulatory. DNase A activity was markedly inhibited by either NaCl or KCl at concentrations greater than 75 mM. The enzyme had a temperature optimum of 25 SC and a pH optimum of about 7 0. Values for K m and K cat were determined to be 61 µM and 330 s N1 respectively, with a catalytic efficiency approximately threefold greater than bovine pancreatic DNase I, but 10-fold less than the Serratia marcescens NucA. DNase A was localized to the periplasm and probably exists as a monomeric species. The enzyme possessed one or more disulfide bonds. In the reduced form it had an apparent mass of 33 kDa, while in the oxidized form it was 29 kDa as estimated by SDS-PAGE. Reduction of the disulfide bonds by dithiothreitol with or without subsequent alkylation by iodoacetamide strongly inactivated the enzyme. DNase A accumulated in vivo had an apparent mass of 29 kDa, indicating that it was in an oxidized form. This is the first indication in a strict anaerobe of a functional periplasmic disulfide bond forming system, phenotypically similar to Dsb systems in facultative and aerobic bacteria.

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Biochemical characterization of Anabaena sp. strain PCC 7120 non‐specific nuclease NucA and its inhibitor NuiA

Cited by 50 publications

References 4 publications

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo

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