Characterization of an amino-terminal fragment of the bacteriophage T4 uvsY recombination protein

D. S. Yassa, K. M. Chou, S. W. Morrical

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22 Scopus citations

Abstract

The uvsY protein plays essential roles in homologous genetic recombination processes in the bacteriophage T4. In vitro, uvsY promotes the formation of presynaptic filaments containing stoichiometric amounts of the T4 uvsX recombinase bound to single-stranded DNA. uvsY protein has intrinsic binding activities towards ssDNA, uvsX, and gp32, the T4-encoded SSB, however, it has not been directly determined which of these activities are essential for uvsY's role in presynapsis. We have therefore sought to generate altered forms of uvsY deficient in uvsX- and/or gp32-binding, in order to assess whether these specific protein-protein interactions are essential for uvsY recombination functions. Limited chymotrypsinolysis of the 16 kDa uvsY protein generates two major fragments: an 11.5 kDa fragment containing the N-terminus of uvsY, and a 4.5 kDa C-terminal fragment. We have expressed and purified the large fragment as a fusion protein containing the N-terminal 101 amino acids of uvsY. We show that this truncated uvsY species, which we call uvsY(NT), retains ssDNA-binding activity, but is devoid of both uvsX- and gp32-binding activities. Like native uvsY, uvsY(NT) stimulates the ssDNA-dependent ATPase activity of the uvsX protein, however, the synergistic effects observed between uvsY, uvsX, and gp32 are not observed with uvsY(NT). In addition, uvsY(NT) weakly stimulates uvsX-catalyzed DNA strand exchange reactions. The latter result is surprising since it suggests that specific interactions with uvsX and/or gp32 are not absolutely essential for uvsY recombination functions. Taken together, the data are consistent with a model in which uvsY-ssDNA interactions alone are capable of promoting the assembly of functional uvsX-ssDNA complexes, while uvsY-protein interactions stabilize uvsX-ssDNA complexes.

Original languageEnglish (US)
Pages (from-to)275-285
Number of pages11
JournalBiochimie
Volume79
Issue number5
DOIs
StatePublished - May 1997

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Keywords

  • Bacteriophage T4
  • Recombination
  • uvsX
  • uvsY

ASJC Scopus subject areas

  • Biochemistry

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