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'-phosphorylated dinucleotide and a phosphodiester not containing a nucleotide residue is not cleaved. Both forms act predominantly in an exonucleolytic 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. Apparent values for the catalytic efficiency of the action on long DNA polymers are too large to fit a simple kinetic scheme involving a direct enzyme-substrate encounter and lead to an interpretation in which nuclease molecules are non-productively bound away from the 5' ends and undergo facilitated diffusion to yield productive (terminally bound) enzyme-substrate complexes.
Lu
T
T
Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934, USA.
Gray
H B
HB
D001345:Autoradiography; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009596:Nitrophenols; D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31