Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa.
Single strand DNA-binding endo-exonucleases purified from mitochondria, vacuoles, or a mixture of these organelles had the same high specific single strand DNase activity (910 mumol of nucleotides/min/mg), and each contained a polypeptide of Mr = 31,000-33,000 which was found to be active by sodium dodecyl sulfate-DNA-gel electrophoresis. The properties of the three preparations were identical in all respects tested. The enzyme showed distributive endonuclease activity with single strand DNA, but processive exonuclease activity with double strand DNA. In the former case, 5'-phosphoryl-terminated fragments were released at early times, while in the latter case, short 5'-oligonucleotides (n = 2-4) were released. Both activities were dependent on Mg2+ (or Mn2+), but to different extents. In 0.1 mM Mg2+, superhelical bacteriophage phi X174 (replicative form (RF II) DNA and, at converted to relaxed circular (RF II) DNA and, at higher enzyme concentrations, to unit length linear (RF III) DNA. In 10 mM Mg2+, these same conversions took place rapidly, and the RF III DNA which formed was degraded to pieces shorter than unit length. At very low enzyme concentrations, long single strand tails and gaps were detected in bacteriophage T7 linear double strand DNA molecules.
D002846:Chromatography, Affinity; D004279:DNA, Viral; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D005092:Exonucleases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D013604:T-Phages; D014617:Vacuoles / Q000201:enzymology
D004279:DNA, Viral; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease