SITE-DIRECTED MUTAGENESIS OF THE CATALYTIC RESIDUES OF BOVINE PANCREATIC DEOXYRIBONUCLEASE-I

Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterise d endonuclease which cleaves double-stranded DNA to yield 5' phosphory lated polynucleotides. Go-crystal structures of DNase I with two diffe rent oligonucleotides have revealed the presence of several residues ( R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. T he roles that these amino acids play in the catalytic mechanism have b een investigated using site-directed mutagenesis. The following varian ts were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics o f all six mutants with both DNA and a small chromophoric substrate, th ymidine-3',5'-di(p-nitrophenyl)-phosphate, were studied. Only R9A and E78T showed. any significant turnover of the two substrates. D168S, H1 34Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3',5'-di-(p-nitrophenyl)-phosph ate. These results demonstrate that H134, D168, D212 and H252 play a c ritical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provid ed general acid and general base catalysis. DNase I also requires Mg2 and E39 has been identified as a ligand for this metal ion. We propos e that D168 serves as a ligand for a second Mg2+, and thus DNase I, us es a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mut agenesis results, structural information, homologies between DNase I f rom different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalyti c role to each amino acid/metal ion. (C) 1996 Academic Press Limited