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

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-HC1 (pH 7.0, 37 degrees 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 NaC1 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.