recA Protein-promoted DNA Strand Exchange Research Articles

Inhibition of RecA Protein Function by the RdgC Protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.

The single-stranded DNA-binding protein of Deinococcus radiodurans

BackgroundDeinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.ResultsA segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.ConclusionsThe correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

A reverse DNA strand exchange mediated by recA protein and exonuclease I. The generation of apparent DNA strand breaks by recA protein is explained

The combined action of exonuclease I and recA protein leads to a kind of reverse DNA strand exchange in which joint molecules formed on the "wrong" or distal end of a linear duplex in the presence of ATP are stabilized by exonuclease I degradation of the displaced (+) strand. Continued pairing and degradation of the displaced strand leads to strand exchange that appears to progress with a polarity opposite that of the normal recA protein promoted reaction (i.e. 3'-5' with respect to the (+) strand). However, in contrast to the normal 5'-3' strand exchange, the displaced strand is completely degraded in the process. When the linear duplex DNA substrate has a heterologous region at the 5' (proximal) end, the major product (described in a previous study (Bedale, W. A., Inman, R. B., and Cox, M. M. (1991) J. Biol. Chem. 266, 6499-6510)) is a circular duplex DNA molecule with a double-stranded tail whose length corresponds closely to the heterologous segment of the substrate. The origin of this product is here shown to be the result of the exonuclease activity of exonuclease I (either added exogenously or present as a trace contaminant of recA protein or SSB protein preparations), as opposed to endonucleolytic or mechanical breakage. The levels of exonuclease I required to generate these products are sufficiently low that they are undetected by assays for exonuclease contamination in recA protein preparations. These results demonstrate that the interplay of recA protein with other enzymes can have a profound effect on both the mechanism and outcome of recA protein-promoted DNA strand exchange. They also demonstrate that the (+) strand of the duplex DNA substrate is at least transiently displaced in recA protein-mediated pairing even when joint molecules are limited to the distal end.

Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents.

To investigate the in vivo effects of macromolecular crowding we examined the effect of inert macromolecules such as polyvinyl alcohol and polyethylene glycol on the in vitro activity of recA protein. The addition of either of these volume-occupying agents enables recA protein to promote homologous pairing and exchange of DNA strands at an otherwise nonpermissive magnesium ion concentration. In the presence of these macromolecules, both the rate of recA protein association with single-stranded DNA (ssDNA) and the steady-state affinity of recA protein for ssDNA are increased. Consequently, the ability of recA protein to compete with ssDNA-binding protein (SSB protein) is enhanced, and the inhibitory effects of SSB protein on the formation of recA protein-ssDNA presynaptic complexes are eliminated. Because the ability of recA protein to bind to ssDNA-containing secondary structures is also enhanced in volume-occupied solution, joint molecule formation is not greatly reduced when SSB protein is omitted from the reaction. Thus, increased recA protein interactions with ssDNA contribute to enhanced presynaptic complex formation. In addition, polyvinyl alcohol and polyethylene glycol must also affect another property of recA protein, i.e. self-association, which is required for synapsis and DNA strand exchange. Our examination of DNA strand exchange in the presence of volume-occupying agents helps to reconcile the requirement for elevated magnesium ion concentrations in recA protein-promoted recombination reactions in vitro, with a presumably low magnesium ion concentration in vivo.

A postsynaptic role for single-stranded DNA-binding protein in recA protein-promoted DNA strand exchange.

The single-stranded DNA-binding protein (SSB protein) is required for efficient genetic recombination in vivo. One function for SSB protein in DNA strand exchange in vitro is to remove secondary structure from single-stranded DNA (ssDNA) and thereby aid in the formation of recA protein-saturated presynaptic complexes. In the preceding paper (Lavery, P. E., and Kowalczykowski, S. C. (1992) J. Biol. Chem. 267, 9307-9314) we demonstrated that DNA strand exchange can occur in the presence of volume-occupying agents at low magnesium ion concentration, where secondary structures are reduced. Our results suggest that SSB protein is not acting during presynapsis under these conditions, yet the DNA strand exchange reaction is stimulated by the addition of SSB protein. In this study we present biochemical evidence which suggests that SSB protein stimulates DNA strand exchange by binding to the ssDNA displaced from joint molecules, thereby stabilizing them and allowing branch migration to extend the region of heteroduplex DNA. Therefore, our results indicate dual roles for SSB protein at elevated magnesium ion concentration; it functions during presynapsis, removing secondary structure from ssDNA, as indicated previously, and it also functions postsynaptically, binding to the ssDNA displaced from joint molecules.

An obligatory pH-mediated isomerization on the [Asn-160]recA protein-promoted DNA strand exchange reaction pathway.

We recently described a mutant recA protein in which glycine 160 of the recA polypeptide was replaced by an asparagine residue (Bryant, F. R. (1988) J. Biol. Chem. 263, 8716-8723). Although the [Asn-160]recA protein has a ssDNA-dependent ATPase activity that is similar to that of the wild-type recA protein, the mutant protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions (pH 7.5, 1 mM ATP). We have found that the [Asn-160]recA protein is able to carry out the three-strand exchange reaction at pH 6.0 to 6.7, but that the strand exchange activity is abolished at higher pH. The induction of strand exchange activity at low pH correlates directly with a pH-mediated activation of an ATP-dependent isomerization of the [Asn-160]recA protein. This ATP-dependent isomerization is characterized by the conversion of the [Asn-160]recA protein to a form that is not displaced from ssDNA by the Escherichia coli SSB protein. In contrast to the pronounced pH sensitivity of the [Asn-160]recA protein, the wild-type recA protein undergoes ATP-dependent isomerization, and is able to carry out the three-strand exchange reaction, over the range of pH 6.0 to 8.4. These results show that the [Asn-160] mutation disrupts the ATP-dependent isomerization of the recA protein and suggest that protonation of the [Asn-160]recA protein (or the [Asn-160]recA-ssDNA complex) relieves this mechanistic defect. Furthermore, the direct correlation between ATP-dependent isomerization and the strand exchange activity of the [Asn-160]recA protein strongly suggests that the ATP-dependent isomerization is an obligatory step in the recA protein-promoted strand exchange mechanism.

Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes.

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.

Stable binding of recA protein to duplex DNA. Unraveling a paradox.

recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.

Exchange of recA protein between adjacent recA protein-single-stranded DNA complexes.

We have examined the exchange of recA protein between stable complexes formed with single-stranded DNA (ssDNA) and (a) other complexes and (b) a pool of free recA protein. We have also examined the relationship of ATP hydrolysis to these exchange reactions. Exchange was observed between two different recA X ssDNA complexes in the presence of ATP. Complete equilibration between two sets of complexes occurred with a t1/2 of 3-7 min under a set of conditions previously found to be optimal for recA protein-promoted DNA strand exchange. Approximately 200 ATPs were hydrolyzed for every detected migration of a recA monomer from one complex to another. This exchange occurred primarily between adjacent complexes, however. Little or no exchange was observed between recA X ssDNA complexes and the free recA protein pool, even after several hundred molecules of ATP had been hydrolyzed for every recA monomer present. ATP hydrolysis is not coupled to complete dissociation or association of recA protein from or with recA X ssDNA complexes under these conditions.

RecA protein-promoted DNA strand exchange. Effect of ionic strength.

The addition of from 80 to 120 mM NaCl to a recA protein-promoted DNA strand exchange results in a significant increase in both the initial rate and extent of reaction. This increase results from the formation of a relatively stable recA protein-single-stranded DNA complex that is similar to the complex formed in the presence of the single-stranded DNA-binding protein. In addition to increasing the rate and extent of strand exchange, the amount of recA protein and ATP hydrolysis needed to promote these reactions efficiently is reduced. However, single-stranded DNA-binding protein is significantly more effective than addition of 100 mM NaCl in promoting stable complex formation and stimulating strand exchange. These findings support the hypothesis that a stable recA protein-single-stranded DNA complex is required for efficient DNA strand exchange.

Unidirectional branch migration promoted by nucleoprotein filaments of RecA protein and DNA.

Described above are the initial results from a series of experiments designed to more carefully define the mechanism of RecA protein-promoted DNA strand exchange, and in particular, the branch migration phase of this reaction. We have also presented two general models for RecA protein-promoted branch migration. Although the data required to demonstrate a particular mechanism are presently unavailable, we believe these models will provide a useful framework to suggest future experiments. The models also suggest possible explanations for observations that are presently unexplained. In particular, it is possible to explain the observed inefficiency of ATP hydrolysis in this system without invoking the notion that much of the ATP hydrolysis observed is simply uncoupled. If the utilization of extensive filaments of RecA protein to carry out branch migration is inherently inefficient, why might such a mechanism be employed by the cell? The answer to this question may lie in the role of RecA protein-promoted branch migration in post-replication repair (West et al. 1981c). It might be expected that the branch points in a RecA protein-promoted branch migration reaction are susceptible to nuclease degradation. Such degradation would halt branch migration and prevent postreplication repair. One of the roles of the RecA nucleoprotein filament may be to protect the branch point from nuclease degradation and ensure formation of an extensive region of heteroduplex DNA. The apparent energetic cost of this reaction may be a relatively small price to pay to ensure the repair of a potentially lethal DNA lesion.

An unpaired 3' terminus stimulates recA protein-promoted DNA strand exchange.

In the presence of 100 mM NaCl, the efficient exchange of strands between a circular single strand and an homologous DNA duplex promoted by the recA and single-stranded DNA binding proteins of Escherichia coli requires an unpaired 3' terminus. Of the duplex DNAs tested, only those with 4 unpaired bases at the 3' termini are effective. Without added NaCl, strand exchange proceeds efficiently with all duplex DNA termini examined including a nicked circular duplex. Thus, at approximately physiological salt concentrations, factors in addition to the recA and single-stranded DNA binding proteins are needed to promote efficient strand exchange. One such factor may be a DNA helicase(s).

On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange.

RecA protein-promoted DNA strand exchange is greatly stimulated by the single-stranded DNA binding protein (SSB) of Escherichia coli. Stimulation is not a consequence of the binding of SSB to excess single-stranded DNA. It results instead from stabilization of recA protein-single-stranded DNA complexes formed in the presence of ATP and SSB. In the presence of SSB, recA protein does not measurably dissociate from these complexes for up to 90 min. However, in its absence, recA protein moves rapidly between two populations of single-stranded DNA, and complete equilibration occurs with t 1/2 of 17 s. Rapid transfer of recA protein to single-stranded DNA occurs during all stages of DNA strand exchange and does not require ATP. The transfer involves an equilibrium between free and bound recA protein rather than a direct redistribution between single-stranded DNA molecules. Thus, SSB prevents dissociation of recA protein from single-stranded DNA, rendering the binding of the recA protein to single-stranded DNA irreversible. Under these conditions, the pairing phase of the strand exchange reaction is accelerated to the point that it is no longer rate-limiting. These results can explain the relative inefficiency of DNA strand exchange in the absence of SSB.

RecA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein.

The recA protein of Escherichia coli promotes the complete exchange of strands between full length linear duplex and single-stranded circular DNA molecules. An early step in this reaction consists of the binding of recA protein to single-stranded DNA. In the presence of ATP and the single-stranded DNA binding protein, recA protein and single-stranded DNA interact to form a complex whose stability depends upon the single-stranded DNA binding protein. Duplex DNA is not required for complex formation. Subsequent steps occur within this complex which contains up to 1 recA protein monomer per 2 nucleotides of single-stranded DNA. Although several hundred ATPs are hydrolyzed per recA protein monomer, recA protein is not released from the complex at any step during or subsequent to strand exchange. The complex is kinetically competent with respect to both its rate of formation and its rate of reaction with homologous duplex DNA. It is therefore a significant intermediate in the overall reaction pathway. These results have served as the basis for an expanded model for recA protein-promoted strand exchange.