論文

査読有り
2014年8月

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

GENES & GENETIC SYSTEMS
  • Wataru Kobayashi
  • ,
  • Satoshi Sekine
  • ,
  • Shinichi Machida
  • ,
  • Hitoshi Kurumizaka

89
4
開始ページ
169
終了ページ
179
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1266/ggs.89.169
出版者・発行元
GENETICS SOC JAPAN

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.

Web of Science ® 被引用回数 : 6

リンク情報
DOI
https://doi.org/10.1266/ggs.89.169
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/25747041
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000350843100003&DestApp=WOS_CPL
ID情報
  • DOI : 10.1266/ggs.89.169
  • ISSN : 1341-7568
  • eISSN : 1880-5779
  • PubMed ID : 25747041
  • Web of Science ID : WOS:000350843100003

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