An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing

Hideki Aihara, Yutaka Ito, Hitoshi Kurumizaka, Tohru Terada, Shigeyuki Yokoyama, Takehiko Shibata

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

RecA protein and its homologs catalyze homologous pairing of dsDNA and ssDNA, a critical reaction in homologous genetic recombination in various organisms from a virus, microbes to higher eukaryotes. In this reaction, RecA protein forms a nucleoprotein filament on ssDNA, which in turn binds to naked dsDNA for homology search. We suggested that the C-terminal domain of RecA protein plays a role in capturing the dsDNA. Here, we isolated the C-terminal domain as a soluble form and determined the solution structure by NMR spectroscopy. The overall folding of the NMR structure agrees with that of the corresponding part of the reported crystal structure, but a remarkable difference was found in a solvent-exposed region due to intermolecular contacts in the crystal. Then, we studied the interaction between the C-terminal domain and DNA, and found that significant chemical shift changes were induced in a specific region by titration with dsDNA. SsDNA induced a much smaller chemical shift perturbation. The difference of DNA concentrations to give the half-saturation of the chemical shift change showed a higher affinity of the C-terminal region toward dsDNA. Combined with our previous results, these provide direct evidence that the defined region in the C-terminal domain furnishes a binding surface for DNA.

Original languageEnglish (US)
Pages (from-to)213-221
Number of pages9
JournalJournal of Molecular Biology
Volume274
Issue number2
DOIs
StatePublished - Nov 28 1997
Externally publishedYes

Bibliographical note

Funding Information:
We are grateful to Dr Taro Nishinaka for helpful discussion. This work was supported by grants for Biodesign Research Program and MR Science Program from RIKEN and from the Ministry of Education, Science and Culture, Japan. H.K. was supported by a fellowship from the Japan Society for the Promotion of Science.

Keywords

  • DNA-binding surface
  • Genetic recombination
  • NMR
  • Solution structure
  • X-ray structure

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