Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase |
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| Authors: | Motoki Takaku Daisuke Takahashi Shinichi Machida Hiroyuki Ueno Noriko Hosoya Shukuko Ikawa Kiyoshi Miyagawa Takehiko Shibata Hitoshi Kurumizaka |
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| Institution: | 1.Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, 2.Laboratory of Molecular Radiology, Center of Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and 3.RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan |
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| Abstract: | The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO IIIα-conjugated beads, and the interaction between EVL and TOPO IIIα was confirmed by a surface plasmon resonance analysis. Purified TOPO IIIα catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO IIIα may function in the processing of DNA intermediates formed during homologous recombination. |
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