Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus.
Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.
Napoli
Alessandra
A
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche Via P. Castellino 111, 80131 Naples, Italy.
Valenti
Anna
A
Salerno
Vincenzo
V
Nadal
Marc
M
Garnier
Florence
F
Rossi
Mosè
M
Ciaramella
Maria
M
D019843:Archaeal Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016944:Sulfolobus / Q000201:enzymology* / Q000378:metabolism; D014466:Ultraviolet Rays
D019843:Archaeal Proteins; D002843:Chromatin; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004247:DNA; C041813:DNA reverse gyrase; D004264:DNA Topoisomerases, Type I