Studies on nuclease alpha from Ustilago maydis.
An endonuclease active upon single-stranded DNA has been extensively purified from Ustilago maydis. The native form of the enzyme appears to be a single polypeptide chain of 55,000 daltons. The enzyme does not require addition of divalent cations for activity, but is stimulated severalfold by the transition metals Co2+, Mn2+, and Zn2+. It is strongly inhibited by ethylenediaminetetraacetic acid and 2-mercaptoethanol. The products of reaction are mononucleotides and small oligonucleotides bearing a 5'-phosphomonoester group. Superhelical DNA is cleaved by the enzyme to an open circular form, then cut to form a linear molecule. Relaxed closed circular duplex DNA is resistant to cleavage. Heteroduplex relaxed circular DNA molecules containing nonhomology or damage in only one strand were constructed and tested as substrates for the enzyme. The rate of enzymatic cleavage was found to be high relative to a control when molecules containing depurinated, deaminated, or radiation damaged sites were tested. Molecules containing a single mismatched base pair were not cleaved any faster than a completely base-paired control, unless reactions were carried out under conditions likely to introduce additional structural distortions in the DNA. Linear duplex DNA exposed to the enzyme is progressively shortened by digestion from both 5' and 3' termini.
D001487:Basidiomycota / Q000201:enzymology*; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D014588:Ustilago / Q000201:enzymology*
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases; C029970:nuclease alpha