Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex

Sunil Nayak, Floyd Ransom Bryant

Research output: Contribution to journalArticle

Abstract

The kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex were examined by stopped-flow fluorescence spectroscopy, using a modified version of the RecA protein that contains a fluorescent reporter group in the ssDNA binding site. The results indicated that: i) an active RecA-ssDNA complex was formed more rapidly on dT200 than on dT50 when either ATP or dATP was provided as the nucleotide cofactor, and ii) active complex formation occurred more rapidly with dATP than with ATP on either dT50 or dT200. The dependence on both the identity of the nucleotide cofactor and the length of the ssDNA effector indicated that active complex formation occurs by a cooperative mechanism and that dATP is more effective than ATP in mediating the interactions between RecA monomers that drive this process. Interestingly, the time courses of dATP-mediated active complex formation were closely similar to those that were obtained with ATPγS, an effectively non-hydrolyzable ATP analog that strongly stabilizes the active conformation of the RecA-ssDNA complex. These results provide mechanistic insight into the enhanced ssDNA binding and DNA strand exchange activities that are observed when dATP is provided in place of ATP in RecA biochemical assays.

Original languageEnglish (US)
Pages (from-to)1257-1261
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume463
Issue number4
DOIs
StatePublished - Aug 7 2015

Fingerprint

Adenosine Triphosphate
Kinetics
Nucleotides
Rec A Recombinases
Fluorescence Spectrometry
Fluorescence spectroscopy
Conformations
Assays
Monomers
Binding Sites
DNA

Keywords

  • Conformational change
  • Nucleotide cofactor
  • RecA protein
  • Stopped-flow fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex. / Nayak, Sunil; Bryant, Floyd Ransom.

In: Biochemical and Biophysical Research Communications, Vol. 463, No. 4, 07.08.2015, p. 1257-1261.

Research output: Contribution to journalArticle

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