Mung bean nuclease cleavage of a dA + dT-rich sequence or an inverted repeat sequence in supercoiled PM2 DNA depends on ionic environment

• Published in: Nucleic acids research 1984-09, Vol.12 (18), p.7087-7104
• Main Authors: Sheflin, Lowell G., Kowalski, David
• Format: Article
• Language: English
• Subjects: Bacteriophages; Base Sequence; DNA Restriction Enzymes - metabolism; DNA, Single-Stranded; DNA, Superhelical; DNA, Viral; Endonucleases - metabolism; Pseudomonas; Repetitive Sequences, Nucleic Acid; Single-Strand Specific DNA and RNA Endonucleases; Substrate Specificity
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/12.18.7087

We have determined the nucleotide sequences around two alternative sites cleaved in supercoiled PM2 DNA by single-strand-specific mung bean nuclease in different ionic environments. In 10 mM Tris-HCl(pH 7.0, 37°C), the major site is a dA+dT-rich sequence which maps with a known early denaturation region at 0.75 map units. About 30 cleavages occurred in a 135 bp region. Cleavages were largely excluded at (dA)n.(dT)n (n=3–7) sequences. Cleavage patterns of this type have not been previously observed in dA+dT-rich sequences. With the addition of 0.1 M NaCl the major alternative site occurred in a hyphenated inverted repeat sequence 500 bp away (0.70 map units) and did not map to an early denaturation region. One major and 4 minor cleavages occurred in the region between the repeats, suggesting that a hairpin containing at most a 12 bp stem and 10 base loop is recognized. The basis for nuclease recognition of the dA+dT-rich sequence is not clear. The differences in the sequences and cleavage patterns at the alternative sites indicate that their secondary structures differ.