Analysis of the Mechanism of the Serratia Nuclease Using Site-Directed Mutagenesis

Friedhoff, Peter; Kolmes, Bettina; Gimadutdinow, Oleg; Wende, Wolfgang; Krause, Kurt L.; Pingoud, Alfred

doi:10.1093/nar/24.14.2632

Cited by 91 publications

(119 citation statements)

References 42 publications

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“…Both the CJE0566 and CJE1441 protein sequences contained a conserved domain of the NUC superfamily (SMART accession no. SM00477) characteristic for DNA/RNA nonspecific endonucleases (11,12). Moreover, the translated sequences contained a DRGH motif (see Fig.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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“…For example, R.KpnI, the first REase member of the HNH superfamily to be identified, exhibits broad cofactor utilization for DNA cleavage (10,149,150). This utilization is also seen in nonspecific endonucleases belonging to the superfamily, e.g., colicin E7, colicin E9, and Serratia nuclease (151)(152)(153). The broad cofactor preference of the HNH enzymes might confer a fitness advantage to the genomes that harbor them by retaining their activity with different metal ions in vivo.…”

Section: Promiscuity In Cofactor Utilization and Substrate Specificitymentioning

confidence: 99%

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Vasu

Nagaraja

2013

Microbiol Mol Biol Rev

509

462

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SUMMARY Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

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“…Despite the frequent statement that His#*& forms the ' active site ' of DNase IIα [24,[38][39][40][41], there is no evidence for the role of this residue in catalysis. Although many endonucleases do feature active-site histidine residues that are involved directly in enzymic activity [31,[42][43][44], His#*& could exhibit its effect on activity through critical participation in DNA binding, for example. To analyse this possibility, wild-type and H295A mutant proteins were tested for their ability to bind DNA using a method developed for the caspase-activated DNase [31].…”

Section: Figure 7 Revised Protein Structure Of Dnase Iiαmentioning

confidence: 99%

Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

MacLea

Krieser

Eastman

2003

Biochemical Journal

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DNase IIα (EC 3.1.22.1) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3′-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure–activity relationship has been largely unexamined. Recently, we demonstrated that active DNase IIα consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415–421]. The present paper describes further work to examine the elements of DNase IIα protein required for activity. Truncated forms and site-specific mutants were expressed in DNase IIα-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase IIα contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase IIα that lacks the purported active-site His295 can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase IIα protein structure, which is important for three-dimensional structural analysis and for production of DNase IIα as a potential protein therapeutic for cystic fibrosis or other disorders.

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Analysis of the Mechanism of the Serratia Nuclease Using Site-Directed Mutagenesis

Cited by 91 publications

References 42 publications

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

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

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

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