Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

• Kobayashi Wataru

  Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University

• Sekine Satoshi

  Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University

• Machida Shinichi

  Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University

• Kurumizaka Hitoshi

  Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University

Abstract

Green fluorescent protein (GFP), fused to the N or C terminus of a protein of interest, is widely used to monitor the localization and mobility of proteins in cells. RAD51 is an essential protein that functions in mitotic DNA repair and meiotic chromosome segregation by promoting the homologous recombination reaction. A previous genetic study with Arabidopsis thaliana revealed that GFP fused to the C terminus of RAD51 (RAD51-GFP) inhibits mitotic DNA repair, but meiotic homologous recombination remained unaffected. To determine how the C-terminal GFP specifically inhibits mitotic DNA repair by RAD51, we purified rice RAD51A1-GFP and RAD51A2-GFP, and performed biochemical analyses. Interestingly, purified RAD51A1-GFP and RAD51A2-GFP are proficient in DNA binding and ATP hydrolysis. However, nucleoprotein complexes containing single-stranded DNA and RAD51A1-GFP or RAD51A2-GFP are significantly defective in binding to the second DNA molecule (secondary DNA binding), and consequently fail to catalyze homologous pairing. In contrast, RAD51A1-GFP and RAD51A2-GFP efficiently stimulated homologous pairing promoted by the meiosis-specific RAD51 isoform DMC1. These biochemical characteristics are well conserved in human RAD51-GFP. Therefore, GFP fused to the C terminus of RAD51 abolishes the homologous pairing activity of RAD51 by disrupting secondary DNA binding, but does not affect its DMC1-stimulating activity.

Journal

• Genes & Genetic Systems

  Genes & Genetic Systems 89 (4), 169-179, 2014

  The Genetics Society of Japan

Keywords

• GFP
• homologous recombination
• RAD51
• DMC1
• homologous pairing

Details

• CRID: 1390282680450708736
• NII Article ID: 130004750050
• NII Book ID: AA11077421
• DOI: 10.1266/ggs.89.169
• ISSN: 18805779
• ISSN: 13417568
• NDL BIB ID: 026154983
• PubMed: 25747041
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I026154983
• Web Site: https://search.jamas.or.jp/link/ui/2016128429
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed