Separate and Combined Biochemical Activities of the Subunits of a Naturally Split Reverse Gyrase

• Published in: The Journal of biological chemistry Vol. 285; no. 51; pp. 39637 - 39645
• Main Authors: Capp, Christopher, Qian, Yushen, Sage, Harvey, Huber, Harald, Hsieh, Tao-shih
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.12.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - genetics; Adenosine Triphosphate - metabolism; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Archaebacteria; ATPases; Catalytic Domain; DNA and Chromosomes; DNA Renaturase; DNA Topoisomerase; DNA Topoisomerases, Type I - genetics; DNA Topoisomerases, Type I - metabolism; DNA Topology; DNA, Archaeal - genetics; DNA, Archaeal - metabolism; DNA, Superhelical - genetics; DNA, Superhelical - metabolism; DNA-dependent ATPase; Genome Stability; Hyperthermophiles; Nanoarchaeota - enzymology; Nanoarchaeota - genetics; Nucleic Acid Enzymology; Positive Supercoiling; Protein Multimerization - physiology; Hyperthermophiles; DNA-dependent ATPase; DNA Renaturase; Positive Supercoiling; Archaebacteria; DNA Topoisomerase; Genome Stability; Nucleic Acid Enzymology; ATPases; DNA Topology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.173989

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.