Sugar non-specific endonucleases

In a continuing effort to identify ribonucleases that may be involved in mRNA decay in Bacillus subtilis, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3-to-5 exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken. These experiments revealed the presence of a high-molecular-weight nuclease encoded by the yhcR gene that was active in the presence of Ca 2؉ and Mn 2؉ . YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3-monophosphate products, similar to the well-characterized micrococcal nuclease of Staphylococcus aureus. YhcR appears to be located principally in the cell wall and is likely to be a substrate for a B. subtilis sortase. Zymogram analysis suggests that YhcR is the major Ca 2؉ -activated nuclease of B. subtilis. In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named "NYD," for "new YhcR domain."A general model for mRNA decay in prokaryotes has been developed, based on studies of Escherichia coli. Decay appears to proceed by a combination of an initiating endonucleolytic cleavage, executed by RNase E, followed by degradation in the 3Ј-to-5Ј direction by polynucleotide phosphorylase (PNPase) or RNase II (5, 23). The final turnover of mRNA is accomplished by oligoribonuclease (9). An E. coli strain lacking PNPase and RNase II is inviable (7). Thus, it is assumed generally that mRNA turnover is an essential function.The genome sequence of B. subtilis has revealed that several of the major E. coli ribonucleases have no homologues in B. subtilis, including RNase E, RNase II, and oligoribonuclease. We have been pursuing biochemical experiments in an effort to identify ribonucleases in B. subtilis that might be responsible for mRNA decay. These studies have resulted in the identification of genes encoding several 3Ј-to-5Ј exoribonucleases: PNPase (17), RNase R (19), and YhaM (20). Mutant strains deficient in these exoribonucleases, alone or in combination, show several different phenotypes (16,19,20,28). The fact that such strains are viable indicates that one or more B. subtilis RNase activities remain to be discovered. In the search for such an RNase, a broad-specificity nuclease encoded by the yhcR gene has been identified and characterized. MATERIALS AND METHODSBacterial strains and plasmid constructions. The wild-type B. subtilis host was BG1, which is trpC2 thr-5. The RNase triple mutant, which was pnpA rnr yhaM, was described previously (20). To delete the yhcR gene, an internal SalI-SacI fragment (nucleotides [nt] 612 to 2336 of the yhcR coding sequence) was replaced with a SalI-SacI fragment from plasmid pBEST501 that contained a neomycin resistance gene cassette (11). Preparation of B. subtilis growth media and competent B. subtilis cultures was performed as described previously (8). E. coli strain DH5␣ (10) was the host for plasmid constructions.To construct His-tagged YhcR, the yhcR codi...

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“…5C). This was similar to the activity of micrococcal nuclease, which has a pH optimum of 9.0 to 10.0 (22).…”

Section: Cloning Of Yhcr and Characterization Of The Protein Productsupporting

confidence: 70%

“…Micrococcal nuclease is a sugar-nonspecific nuclease (22). To test whether YhcR could cleave single-stranded DNA, an 18-nt, 5Ј-end-labeled oligodeoxyribonucleotide was incubated with purified YhcR.…”

Section: Cloning Of Yhcr and Characterization Of The Protein Productmentioning

confidence: 99%

Bacillus subtilis YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure

Oussenko

Sánchez

Bechhofer³

2004

J Bacteriol

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“…Moreover, the translated sequences contained a DRGH motif (see Fig. S1 in the supplemental material) characteristic for the active site of members of the Serratia nuclease subfamily (26). Predicted structural models of CJE0566 and CJE1441 showed similarities with the crystal structure of NucA from Anabaena sp.…”

Section: Resultsmentioning

confidence: 93%

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

et al. 2010

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The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in the absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophageencoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA.

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“…Though pure nucleic acids are generally not sufficient as a sole carbon source for bacteria, the soil bacterium Serratia marcescens and the intestinal bacterium E. coli are capable of utilizing DNA exclusively for carbon (Beliaeva et al, 1976;Benedik and Strych, 1998;Finkel and Kolter, 2001). Depending on their mode of action, nucleases that degrade DNA substrates are classified as sugar-specific deoxyribonucleases (exo-deoxyribonucleases, endo-deoxyribonucleases and restriction endonucleases), or sugar non-specific nucleases (endonucleases and exonucleases) (Rangarajan and Shankar, 2001). Both single-and double-stranded nucleases are widespread (Desai and Shankar, 2003).…”

Section: Enzymatic Degradation Of Dnamentioning

confidence: 99%

Release and persistence of extracellular DNA in the environment

Nielsen

Johnsen

Bensasson

et al. 2007

Environ. Biosafety Res.

484

382

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The introduction of genetically modified organisms (GMOs) has called for an improved understanding of the fate of DNA in various environments, because extracellular DNA may also be important for transferring genetic information between individuals and species. Accumulating nucleotide sequence data suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in bacterial evolution. The uptake of extracellular DNA by natural transformation is one of several ways bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA. We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment and water, with a focus on the accessibility of the extracellular nucleic acids as substrate for competent bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular DNA are presented. There is a need for improved methods for accurately determining the degradation routes and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms throughout their lifecycles.

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Sugar non-specific endonucleases

Cited by 23 publications

References 151 publications

Bacillus subtilis YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure

Bacillus subtilis YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure

Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

Release and persistence of extracellular DNA in the environment

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