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

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 singlestranded 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.The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in vitro DNA strand exchange reaction between single-stranded (ssDNA) 1 and homologous double-stranded DNA (dsDNA) molecules. The DNA strand exchange reaction takes place in several stages (Fig. 1). The RecA protein forms a nucleoprotein filament that completely encompasses the circular ssDNA. This filament then aligns the bound single strand with a homologous duplex DNA to form a DNA pairing intermediate often referred to as a joint molecule. 1000 base pairs of DNA can be aligned and exchanged in a joint molecule under the empirically defined optimal reaction conditions, which typically include 1-3 mM ATP and about 10 mM magnesium ion. All steps to this point, including the formation of joint molecules, require ATP but not ATP hydrolysis. ATP hydrolysis is needed only to complete the late stages of strand exchange of long DNA substrates, often derived from bacteriophage DNAs. Whereas DNA pairing, leading to joint molecule formation, can occur at either end of a linear duplex, the subsequent and ATP hydrolysisdependent extension of the nascently paired regions is unidirectional, proceeding 5Ј to 3Ј relative to ssDNA initially bound in the filament. Thus, exchange proceeds in one direction along the linear duplex, and joints formed at the "wrong" end in the pairing phase are eliminated. In a DNA strand exchange involving quite long DNAs that leads to nicked circular product formation, the ends of the duplex where the exchange begins and ends are referred to as proximal and distal, respectively ( Fig. 1) (1, 2).Examination of the conditions for an optimal RecA prot...

The C Terminus of the Escherichia coli RecA Protein Modulates the DNA Binding Competition with Single-stranded DNA-binding Protein

“…SSB has multiple salt-dependent DNA binding modes (30,59) interaction site or sites on RecA protein outside of the C terminus that affect the capacity of RecA to displace SSB. In contrast, an interaction of Mg 2ϩ with these sites does not appear to be required for the strand exchange reaction, since the excess Mg 2ϩ requirement in that reaction was largely eliminated for RecA⌬C17 (44).…”

“…The truncated proteins dramatically alter the pH-reaction profile for DNA strand exchange (43) and exhibit a progressive reduction in the requirement for free Mg 2ϩ in the same reaction (44). For the RecA⌬C17 mutant protein, there is no measurable requirement for Mg 2ϩ in excess of that required to coordinate the ATP used in a given experiment (44). At Mg 2ϩ concentrations above their respective optima, reactions promoted by the mutant proteins are somewhat inhibited and produce DNA species that do not migrate into the gel.…”

“…To more systematically explore the function of the C terminus, C-terminal deletions removing 6, 13, and 17 amino acids were constructed and characterized in detail (43,44). The truncated proteins dramatically alter the pH-reaction profile for DNA strand exchange (43) and exhibit a progressive reduction in the requirement for free Mg 2ϩ in the same reaction (44).…”

RecFOR and RecOR as Distinct RecA Loading Pathways

“…The LexA repressor protein controls expression of the recA gene (15,16). The 17 C-terminal amino acids of RecA function in autoregulation of most protein activities (17)(18)(19). Finally, E. coli possesses many additional proteins that function as recombination regulators, with RecA as their target (20 -22).…”

A model for the RecA protein of Mycoplasma synoviae

“…In addition, the initiation proteins (such as RecBCD and RecFOR) might interact directly with the RecA protein during the filament nucleation process, altering RecA conformation so that the C-terminus is no longer inhibitory. These data sets suggest that the DNA binding regulation ability of RecA and/or its ability to compete with SSB would allow RecA to gain access to SSB-coated DNA only at the appropriate time, such as after a replication fork stalls, and that the C-terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament, thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations (Eggler et al, 2003;Lusetti et al, 2003aLusetti et al, , 2003b. In MsRecA, the absence of the last 25 amino acids from the C-terminal domain (a region rich in negatively charged amino acids) could have implications for its functions since regulatory mechanisms, such as those described for EcRecA, can be affected.…”