Abstract
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.
Highlights
We have purified an acid-soluble DNA endonuclease, termed nuclease y, from Ustilago cellextracts
Divalent cations direct the mode by which the enzyme cleaves duplex DNA
We first detected nuclease y as a DNA endonuclease contaminating preparations of highmobilitygroupchromosomal proteins from Ustilago (Rowe, 1982).Wepurified the enzyme and found that it introducesdingle or double strand cuts inDNA depending on the divalent cation present
Summary
From the Departmentof Immunology and Medical Microbiology, Uniuersity of Florida College of Medicine, Gainesuille, Florida 32610. We have purified an acid-soluble DNA endonuclease, termed nuclease y, from Ustilago cellextracts. Divalent cations direct the mode by which the enzyme cleaves duplex DNA. Nuclease 01, is an endonuclease of M , = 55,000, possibly a metalloprotein, highly active on single-stranded DNA (Holloman etal., 1981). The other enzyme, nuclease p, is highly active onsingle-stranded DNA butcan be distinguished from nucleasecy by its higher M , = 68,000, its activity in the presence of EDTA, and its generation of nucleotide residues terminating in3’-phosphomonoester groups in DNA digests (Rusche et al, 1980). We first detected nuclease y as a DNA endonuclease contaminating preparations of highmobilitygroupchromosomal proteins from Ustilago (Rowe, 1982).Wepurified the enzyme and found that it introducesdingle or double strand cuts inDNA depending on the divalent cation present.
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