A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction

Jin Inoue, Takayuki Nagae, Masaki Mishima, Yutaka Ito, Takehiko Shibata, Tsutomu Mikawa

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.

Original languageEnglish
Pages (from-to)6720-6732
Number of pages13
JournalJournal of Biological Chemistry
Volume286
Issue number8
DOIs
Publication statusPublished - 2011 Feb 25

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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