Cooperativity and Intermediate Structures of Single-stranded DNA Binding–Assisted RecA-Single-Stranded DNA Complex Formation Studied by Atomic Force Microscopy

K. Umemura,* T. Okada,* R. Kuroda*,†,‡

*Joint Research Center for Atom Technology, Ibaraki; †Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo; and ‡Kuroda Chiromorphology Team, JST ERATO-SORST, Tokyo, Japan

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Summary: The formation of a complex between RecA protein and single-stranded (ss) DNA was studied systematically by atomic force microscopy (AFM) by varying incubation time and the molecular ratio of RecA protein to single-stranded DNA binding (SSB) protein. New intermediate structures, such as small circular, tangled, and protruded structures in the absence of SSB and sharply turned structures in the presence of SSB, were clearly identified at the early stage of complex formation. These structures have probably resulted from competitive binding of RecA and SSB to DNA. After long incubation, only fully covered RecA-ssDNA and totally RecA-free SSB-ssDNA complexes were present regardless of RecA concentrations. Together with intermediate structures which consisted of only two parts, that is, ssDNA covered by SSB and by RecA proteins, the observation suggested strong neighbor cooperative binding of RecA to ssDNA assisted by SSB.

Key words: RecA, single-stranded DNA binding protein, single-stranded (ss) DNA, atomic force microscopy

PACS: 87.64.Dz

This work was performed under the management of the Atom Technology Partnership (ATP) in the Joint Research Center for Atom Technology (JRCAT), and partly supported by the New Energy and Industrial Technology Development Organization (NEDO).