title: Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair.
abstract: In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.
authors.affiliation: Cellular & Molecular Biology Unit, RIKEN, 2-1 Hirosawa, 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.forename: Takeshi
authors.initials: T
authors.lastname: Shinohara
authors.affiliation: Cellular & Molecular Biology Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Advanced Catalysis Research Group, RIKEN Center for Sustainable Resource Science, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Shukuko
authors.initials: S
authors.lastname: Ikawa
authors.affiliation: Cellular & Molecular Biology Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Wakana
authors.initials: W
authors.lastname: Iwasaki
authors.affiliation: Advanced Photon Technology Division, Research Infrastructure Group, RIKEN SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Hiraki
authors.affiliation: Advanced Photon Technology Division, Research Infrastructure Group, RIKEN SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hikima
authors.affiliation: Cellular & Molecular Biology Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Mikawa
authors.affiliation: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, 1866 Kameino, Fujisawa-shi, Kanagawa 252-8510, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Arai
authors.affiliation: Advanced Photon Technology Division, Research Infrastructure Group, RIKEN SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Kamiya
authors.affiliation: Cellular & Molecular Biology Unit, RIKEN, 2-1 Hirosawa, 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 tshibata@riken.jp.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shibata
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000378:metabolism; D055162:Biocatalysis; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D059765:Homologous Recombination; D011485:Protein Binding; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D000099160:LexA Repressor Protein
chemical_list: D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D004247:DNA; D004277:DNA, Single-Stranded; D011938:Rec A Recombinases; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein