Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Shelley L Lusetti,Jeffrey J Shaw,Michael M Cox

doi:10.1074/jbc.m212916200

Shelley L Lusetti, Jeffrey J Shaw + Show 1 more

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https://doi.org/10.1074/jbc.m212916200

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Abstract

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

Highlights

The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair
We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations
RecA⌬C17 Protein Is Unable to Promote Complete DNA Strand Exchange Reactions with ATP or Promote Joint Molecule Formation with ATP␥S at pH 6, Even at Low Magnesium Ion Concentrations—In an accompanying paper [23] characterizing the various C-terminal deletion mutants of the RecA protein, we showed that the mutants were deficient in their ability to generate nicked circular products in DNA strand

Summary

Introduction

The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair. The RecA protein forms a nucleoprotein filament that completely encompasses the circular ssDNA This filament aligns the bound single strand with a homologous duplex DNA to form a DNA pairing intermediate often referred to as a joint molecule. A proposal was advanced that the effect could be attributed to the elimination of electrostatic repulsion between the negatively charged residues in the C terminus and the phosphates in the DNA [17, 18] Both C-terminal deletion mutants promoted DNA strand exchange under at least some conditions, and the larger deletion exhibited an enhanced DNA strand exchange in the absence of single-stranded DNA-binding protein (SSB) [17, 18]. A 25-residue C-terminal deletion of the RecA protein of Proteus mirabilis exhibited improved binding to dsDNA [19]

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