The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks.

RecN is a cohesin-like protein involved in DNA double-strand break repair in bacteria. The RecA recombinase functions to mediate repair via homologous DNA strand invasion to form D-loops. Here we provide evidence that the RecN protein stimulates the DNA strand invasion step of RecA-mediated recombinational DNA repair. The intermolecular DNA tethering activity of RecN protein described previously cannot fully explain this novel activity since stimulation of RecA function is species-specific and requires RecN ATP hydrolysis. Further, DNA-bound RecA protein increases the rate of ATP hydrolysis catalysed by RecN during the DNA pairing reaction. DNA-dependent RecN ATPase kinetics are affected by RecA protein in a manner suggesting a specific order of protein-DNA assembly, with RecN acting after RecA binds DNA. We present a model for RecN function that includes presynaptic stimulation of the bacterial repair pathway perhaps by contributing to the RecA homology search before ternary complex formation.

The Escherichia coli DinD protein modulates RecA activity by inhibiting postsynaptic RecA filaments.

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to single-stranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.

RecN is a cohesin-like protein that stimulates intermolecular DNA interactions in vitro.

The bacterial RecN protein is involved in the recombinational repair of DNA double-stranded breaks, and recN mutants are sensitive to DNA-damaging agents. Little is known about the biochemical function of RecN. Protein sequence analysis suggests that RecN is related to the SMC (structural maintenance of chromosomes) family of proteins, predicting globular N- and C-terminal domains connected by an extensive coil-coiled domain. The N- and C-domains contain the nucleotide-binding sequences Walker A and Walker B, respectively. We have purified the RecN protein from Deinococcus radiodurans and characterized its DNA-dependent and DNA-independent ATPase activity. The RecN protein hydrolyzes ATP with a k(cat) of 24 min(-1), and this rate is stimulated 4-fold by duplex DNA but not by single-stranded DNA. This DNA-dependent ATP turnover rate exhibits a dependence on the concentration of RecN protein, suggesting that RecN-RecN interactions are required for efficient ATP hydrolysis, and those interactions are stabilized only by duplex DNA. Finally, we show that RecN stimulates the intermolecular ligation of linear DNA molecules in the presence of DNA ligase. This DNA bridging activity is strikingly similar to that of the cohesin complex, an SMC family member, to which RecN is related.

Disassembly of Escherichia coli RecA E38K/DeltaC17 nucleoprotein filaments is required to complete DNA strand exchange.

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/DeltaC17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/DeltaC17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/DeltaC17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.

The RecF protein antagonizes RecX function via direct interaction.

The RecX protein inhibits RecA filament extension, leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto single-stranded DNA binding protein (SSB)-coated circular single-stranded DNA (ssDNA) in the presence of RecX occurs only when all of the RecFOR proteins are present. The RecOR proteins contribute only to RecA filament nucleation onto SSB-coated single-stranded DNA and are unable to counter the inhibitory effects of RecX on RecA filaments. RecF protein uniquely supports substantial RecA filament extension in the presence of RecX. In vivo, RecF protein counters a RecX-mediated inhibition of plasmid recombination. Thus, a significant positive contribution of RecF to RecA filament assembly is to antagonize the effects of the negative modulator RecX, specifically during the extension phase.

The DinI and RecX proteins are competing modulators of RecA function.

The DinI and RecX proteins of Escherichia coli both modulate the function of RecA protein, but have very different effects. DinI protein stabilizes RecA filaments, preventing disassembly but permitting assembly. RecX protein blocks RecA filament extension, which can lead to net filament disassembly. We demonstrate that both proteins can interact with the RecA filament, and propose that each can replace the other. The DinI/RecX displacement reactions are slow, requiring multiple minutes even when a large excess of the challenging protein is present. The effects of RecX protein on RecA filaments are manifest at lower modulator concentrations than the effects of DinI protein. Together, the DinI and RecX proteins constitute a new regulatory network. The two proteins compete directly as mainly positive (DinI) and negative (RecX) modulators of RecA function.

Inhibition of RecA protein by the Escherichia coli RecX protein: modulation by the RecA C terminus and filament functional state.

The RecX protein is a potent inhibitor of RecA activities. We identified several factors that affect RecX-RecA interaction. The interaction is enhanced by the RecA C terminus and by significant concentrations of free Mg(2+) ion. The interaction is also enhanced by an N-terminal His(6) tag on the RecX protein. We conclude that RecX protein interacts most effectively with a RecA functional state designated A(o) and that the RecA C terminus has a role in modulating the interaction. We further identified a C-terminal point mutation in RecA protein (E343K) that significantly alters the interaction between RecA and RecX proteins.

A RecA filament capping mechanism for RecX protein.

The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.

The DinI protein stabilizes RecA protein filaments.

When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA. Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods. DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities. Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected. Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration. DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed. The interaction between the RecA protein and DinI was modulated by the C terminus of RecA. We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.

The C terminus of the Escherichia coli RecA protein modulates the DNA binding competition with single-stranded DNA-binding protein.

The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.

C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects.

A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK(a) of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

Magnesium ion-dependent activation of the RecA protein involves the C terminus.

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.

The bacterial RecA protein and the recombinational DNA repair of stalled replication forks.

The primary function of bacterial recombination systems is the nonmutagenic repair of stalled or collapsed replication forks. The RecA protein plays a central role in these repair pathways, and its biochemistry must be considered in this context. RecA protein promotes DNA strand exchange, a reaction that contributes to fork regression and DNA end invasion steps. RecA protein activities, especially formation and disassembly of its filaments, affect many additional steps. So far, Escherichia coli RecA appears to be unique among its nearly ubiquitous family of homologous proteins in that it possesses a motorlike activity that can couple the branch movement in DNA strand exchange to ATP hydrolysis. RecA is also a multifunctional protein, serving in different biochemical roles for recombinational processes, SOS induction, and mutagenic lesion bypass. New biochemical and structural information highlights both the similarities and distinctions between RecA and its homologs. Increasingly, those differences can be rationalized in terms of biological function.