Biochemical activities of denatured covalently closed circular duplex DNA.

Covalently closed circular duplex DNA when exposed to high alkaline pH followed by neutralization yields a collapsed state structure (form I(d)) that can undergo transition to form I and was susceptible to S1 nuclease. Form I(d), in spite of its compact structure, admits specific cleavage by restriction enzymes over its entire genome. When used in a semi-in vitro replication complex, form I(d) gave significantly better template activity, and undergoes better primer extension in in vitro using Klenow. Thus form I(d) having a compact shape can behave as a better substrate in a few key enzymatic reactions.