Champoux J J
Proc Natl Acad Sci U S A. 1977 Sep;74(9):3800-4. doi: 10.1073/pnas.74.9.3800.
Strands of DNA that have been broken by the DNA untwisting enzyme exhibit a reduced buoyant density in alkaline CsCl due to bound protein. A covalent linkage between the DNA and the enzyme was indicated by the stability of the complex in alkali (pH greaterthan 12.7), in 7 M guanidine-HCl, and at 90 degrees in 1% Sarkosyl for 5 min. The single-strand breaks generated by the enzyme are resistant to exonuclease III, indicating that the protein is attached to one of the ends of the broken strands. The free end of the broken strand bears a 5'-hydroxyl group, indicating attachment of the protein to the 3'-phosphoryl terminus. A nucleotide-peptide linkage involving a phosphoamide bond is unlikely since the complex is resistant to 3.5 M hydroxylamine at pH 4.75.
被DNA解旋酶切断的DNA链由于结合了蛋白质，在碱性氯化铯中表现出降低的浮力密度。DNA与酶之间的共价连接通过复合物在碱（pH大于12.7）、7M盐酸胍以及在1%十二烷基肌氨酸钠中90℃处理5分钟后的稳定性得以表明。该酶产生的单链断裂对外切核酸酶III具有抗性，这表明蛋白质附着在断裂链的一端。断裂链的自由端带有一个5'-羟基，表明蛋白质附着在3'-磷酸末端。由于复合物在pH 4.75的3.5M羟胺中具有抗性，所以涉及磷酰胺键的核苷酸-肽连接不太可能。
