Purification and properties of nuclease gamma from Ustilago maydis.
We have purified an acid-soluble DNA endonuclease, termed nuclease gamma, from Ustilago cell extracts. The enzyme is nearly homogeneous, purified 1700-fold. The protein appears to be globular with a molecular weight in the range 17,000 to 21,000. It requires a divalent cation and is optimally active at slightly alkaline pH. The enzyme prefers duplex DNA as substrate but will slowly cleave single-stranded DNA. Cleavage of covalently closed duplex DNA is unaltered by changes in superhelix density. Divalent cations direct the mode by which the enzyme cleaves duplex DNA. When Mg2+ or Ca2+ is added, the enzyme nicks one strand of the duplex. When Mn2+, Co2+, or Zn2+ is added, the enzyme can introduce double strand breaks. Oligonucleotides terminated with 5'-phosphoryl and 3'-hydroxyl groups are the products of hydrolysis. DNA fragments generated can be religated to linear pBR322 DNA with completely base-paired ends.
D001487:Basidiomycota / Q000201:enzymology*; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification*; D005656:Fungal Proteins; D008970:Molecular Weight; D013379:Substrate Specificity; D014588:Ustilago / Q000201:enzymology*
D002413:Cations, Divalent; D005656:Fungal Proteins; D004706:Endodeoxyribonucleases; C040781:nuclease gamma, Ustilago maydis