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Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex |
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| Authors: | Mayanagi Kouta Fujiwara Yoshie Miyata Tomoko Morikawa Kosuke |
| Institution: | a Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan b Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan c Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan d The Takara-Bio Endowed Division, Institute for Protein Research, Osaka University, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan e BIRD, JST, Japan f CREST, JST, Japan |
| Abstract: | During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB. |
| Keywords: | Computer assisted image processing DNA binding proteins Electron microscopy Holliday junction Homologous recombination Resolvase RuvAB Single particle analysis Thermus thermophilus |
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