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Effect of divalent ions on the minimal relaxase domain of MobA

Shuangluo Xia1, Jon D Robertus

  • 1Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.

Archives of Biochemistry and Biophysics
|June 17, 2009
PubMed

Insights

Divalent metal cations are essential for the DNA nicking activity of the minimal MobA protein (minMobA) but not for its DNA binding. Magnesium (Mg2+) and calcium (Ca2+) ions facilitate catalysis, suggesting a structural role for cations in bacterial conjugation.

Area of Science:

  • Bacteriology
  • Molecular Biology
  • Biochemistry

Background:

  • The MobA protein from plasmid R1162 is crucial for bacterial conjugation.
  • MobA possesses distinct N-terminal relaxase and C-terminal primase domains.
  • The N-terminal 186 residues (minMobA) constitute the minimal functional unit for relaxase activity.

Purpose of the Study:

  • To investigate the influence of various divalent metallic cations on the DNA binding and nicking activities of minMobA.
  • To elucidate the structural and catalytic roles of divalent cations in the relaxase function of MobA.

Main Methods:

  • In vitro experiments were conducted to assess minMobA's DNA binding capabilities.
  • DNA nicking assays were performed to measure enzymatic activity in the presence of different cations.
  • Protein denaturation experiments were utilized to evaluate cation-induced structural changes.

Main Results:

  • Divalent cations are not required for minMobA's DNA binding.
  • DNA nicking activity is dependent on the presence of specific divalent cations.
  • Mn(2+) showed the strongest binding, while Mg(2+) and Ca(2+) effectively facilitated catalysis.
  • Zn(2+) did not support DNA binding or nicking activity, indicating specificity.

Conclusions:

  • The primary role of divalent cations in minMobA activity is structural, influencing catalysis rather than initial DNA recognition.
  • Mg(2+) and Ca(2+) are key cofactors for the relaxase function of minMobA.
  • The differential effects of various cations highlight the intricate mechanism of bacterial DNA mobilization.

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