The DNA RNA non-specific Serratia nuclease prefers double-stranded A-form nucleic acids as substrates

A steady-state kinetic analysis of the cleavage of the oligonucleotides d(C GCTTTTTTGC) (d(y)), d(GCAAAAAAGCG) (d(r)), r(CGCUUUUUUGC) (r(y)) and r(GCAA AAAAGCG) (r(r)) in single and double-stranded form by the extracellular Ser ratia marcescens endonuclease, in conjunction with structural data from a c ircular dichroism spectroscopic analysis of these substrates, suggests that oligonucleotides adopting the A-conformation are preferred over those adop ting the B-conformation as substrates. Relative catalytic efficiencies (k(c at)/K-M) 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 pur ine-rich single-stranded oligonucleotides r(r) and d(r), are cleaved more e fficiently than the pyrimidine-rich oligonucleotides, r(y) and d(y), presum ably because they adopt helical structures with pronounced base stacking. E xcept for the double-stranded oligodeoxynucleotide substrate, the individua l strands are cleaved more efficiently when incorporated into a duplex, tha n in a single-stranded form. Cleavage experiments with various polynucleoti des, including a viroid RNA and a specifically designed 167bp DNA, confirm that double-stranded A-form nucleic acids are preferentially attacked by Se rratia nuclease. In an attempt to analyze the basis of these preferences, w e have mutated the amino acid residues Tyr76 and Trp123 of Serratia nucleas e. 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 base s, which could lead to the cleavage preferences observed. However, only eff ects on the activity, but no change of the sequence or substrate preference s, were detected upon substitution of these amino acid residues, ruling out any involvement of these residues in the A-form preference of Serratia nuc lease. (C) 1999 Academic Press.