Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease

Abstract

The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serrutia nuclease. Most of the natural substrates are cleaved with similar k,,, and K,,, values, the k,,,lK,,, ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA) . poly(dT) or poly(dG) . poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K,,,, respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-R,-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the R,-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the R,-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p4trophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-@-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

“…When Mg 2+ was replaced by Ca 2+ in the reaction mix, no endonuclease activity was detected. The data indicate that CuquEndo is divalent cation dependent, similar to S.·marcescens endonuclease, which requires Mg 2+ ions, but it is insensitive to Ca 2+ ions (Friedhoff et al, 1996).…”

“…The active center of S.·marcescens endonuclease has the invariable residues His89, Arg57, Asn106, Asn119 and Glu127, which are also present in equivalent positions in the CuquEndo putative active center (Fig.·1). The importance of these residues has been demonstrated by mutagenesis of every one of them, resulting in a drastic reduction in the catalytic properties of S. marcescens endonuclease (Friedhoff et al, 1996). His89 of S. marcescens (CuquEndoHis171) nuclease and the magnesium water cluster are directly involved in the phosphodiester bond cleavage of the DNA substrate.…”

“…Plasmid DNA was cleaved to fragments with retention time ranging from 17 to 22·min (5-10·nt compared with polyT standard). Similarly, Friedhoff et al reported (Friedhoff et al, 1996) that for moderately good cleavage activity by the Serratia nuclease, the deoxynucleotide substrate should contain at least five phosphate residues. Results of incubation of synthetic ss and ds deoxynucleotides in the presence of recombinant CuquEndo (Fig.·7) clearly indicate that this enzyme only cleaves dsDNA.…”

“…Our interpretation is that fragments shorter than five are ss at the temperature (37°C) at which we carried out the enzymatic assays and thus cannot be cleaved by CuquEndo, which appears to be a dsspecific endonuclease. Other nonspecific endonucleases exhibit strong cleavage preference for dsDNA but, unlike CuquEndo, low levels of hydrolytic activity toward ssDNA have been reported (Friedhoff et al, 1996;Shagin et al, 2002;Wang et al, 2000). Starved mosquitoes were allowed to probe in an agarose gel containing 10·mmol·l -1 NaHCO 3 , 100·mmol·l -1 NaCl, 1·mmol·l…”

“…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.…”