title: The DNA/RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates.
abstract: A steady-state kinetic analysis of the cleavage of the oligonucleotides d(CGCTTTTTTGC) (d(y)), d(GCAAAAAAGCG) (d(r)), r(CGCUUUUUUGC) (r(y)) and r(GCAAAAAAGCG) (r(r)) in single and double-stranded form by the extracellular Serratia marcescens endonuclease, in conjunction with structural data from a circular dichroism spectroscopic analysis of these substrates, suggests that oligonucleotides adopting the A-conformation are preferred over those adopting the B-conformation as substrates. Relative catalytic efficiencies (kcat/KM) for the cleavage of the homo- and heteroduplexes follow the order r(r).r(y) (1.0)>r(r).d(y) (0.9)>d(r). r(y) (0.7)>d(r).d(y) (0.3). The purine-rich single-stranded oligonucleotides r(r) and d(r), are cleaved more efficiently than the pyrimidine-rich oligonucleotides, r(y) and d(y), presumably because they adopt helical structures with pronounced base stacking. Except for the double-stranded oligodeoxynucleotide substrate, the individual strands are cleaved more efficiently when incorporated into a duplex, than in a single-stranded form. Cleavage experiments with various polynucleotides, including a viroid RNA and a specifically designed 167 bp DNA, confirm that double-stranded A-form nucleic acids are preferentially attacked by Serratia nuclease. In an attempt to analyze the basis of these preferences, we have mutated the amino acid residues Tyr76 and Trp123 of Serratia nuclease. These residues are located close to the active site and are conserved in all members of the Serratia nuclease family, suggesting that they could be involved in substrate binding, e.g. by stacking interactions with the bases, which could lead to the cleavage preferences observed. However, only effects on the activity, but no change of the sequence or substrate preferences, were detected upon substitution of these amino acid residues, ruling out any involvement of these residues in the A-form preference of Serratia nuclease.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010942:Plant Viruses / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014772:Viroids / Q000235:genetics