title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.
abstract: RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.
authors: Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan.|Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.|Department of Supramolecular Biology, Graduate School of Nanobiosciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors: Naoto
authors: N
authors: Konomura
authors: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa 252-0880, Japan narai@brs.nihon-u.ac.jp.
authors: Naoto
authors: N
authors: Arai
authors: Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan.|Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.|Department of Supramolecular Biology, Graduate School of Nanobiosciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors: Takeshi
authors: T
authors: Shinohara
authors: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa 252-0880, Japan.
authors: Jun
authors: J
authors: Kobayashi
authors: Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors: Wakana
authors: W
authors: Iwasaki
authors: Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan.|Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.
authors: Shukuko
authors: S
authors: Ikawa
authors: Center for Genetic Resource Education & Development, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
authors: Kohji
authors: K
authors: Kusano
authors: Cellular & Molecular Biology Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japan tshibata@riken.jp.|Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.|Department of Supramolecular Biology, Graduate School of Nanobiosciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors: Takehiko
authors: T
authors: Shibata
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D003067:Coenzymes; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D011938:Rec A Recombinases