KINETICS AND MECHANISM OF BAL-31 NUCLEASE ACTION ON SMALL SUBSTRATES AND SINGLE-STRANDED-DNA

Kinetic and mechanistic aspects of the action of two forms of the BAL 31 nuclease (EC 3.1.11) from Alteromonas espejiana on model substrates , small oligonucleotides, larger oligonucleotides and poly[d(A)] have been examined. The minimal oligonucleotide substrate is a 5'-phosphory lated dinucleotide and a phosphodiester not containing a nucleotide re sidue is not cleaved. Both forms act predominantly in an exonudeolytic fashion on single-stranded DNA polymers in a highly processive manner ; however, the mechanism becomes distributive for small oligomers (3-4 nucleotide residues). The direction of attack is from the 5' end, in contrast to the mode of digestion of duplex DNA which involves attack at the 3' termini. An endonucleolytic mode of attack also exists, but at a level 2-3% or less of that of the terminally directed cleavage. A pparent values for the catalytic efficiency of the action on long DNA polymers are too large to fit a simple kinetic scheme involving a dire ct, enzyme-substrate encounter and lead to an interpretation in which nuclease molecules are non-productively bound away from the 5' ends an d undergo facilitated diffusion to yield productive (terminally bound) enzyme-substrate complexes.