Combinatorial analyses including enzymatic analysis and atomic force microscopy (AFM) were used to gain more insights into structure of the alkali-denatured plasmid pBR322 DNA (DNA Ⅳ). Among the restriction enzymes tested, PstⅠ was able to cleave the pBR322 DNA Ⅳ molecules, indicative of the existence of an intact restriction site in the denatured DNA. AFM images revealed that PstⅠ-treated DNA Ⅳ molecules contained an individual node. The contour length of the condensed molecules was decreased by about 11% compared to DNA Ⅳ. Intriguingly, the Escherichia coli topoisomerase Ⅳ, a type Ⅱ topoisomerase, also introduced nodes into DNA Ⅳ while keeping the molecules in a closed form. The two types of DNA were similar to each other in terms of the contour length and the size of the node based on the AFM analysis. These findings suggest that specific base-paired regions exist in DNA Ⅳ, and that cleavages at these regions may lead to DNA node.