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 recombination-associated protein RdgC adopts a novel toroidal architecture for DNA binding

RecA plays a central role in the nonmutagenic repair of stalled replication forks in bacteria. RdgC, a recombination-associated DNA-binding protein, is a potential negative regulator of RecA function. Here, we have determined the crystal structure of RdgC from Pseudomonas aeruginosa. The J-shaped monomer has a unique fold and can be divided into three structural domains: tip domain, center domain and base domain. Two such monomers dimerize to form a ring-shaped molecule of approximate 2-fold symmetry. Of the two inter-subunit interfaces within the dimer, one interface (‘interface A’) between tip/center domains is more nonpolar than the other (‘interface B’) between base domains. The structure allows us to propose that the RdgC dimer binds dsDNA through the central hole of ~30Å diameter. The proposed model is supported by our DNA-binding assays coupled with mutagenesis, which indicate that the conserved positively charged residues on the protein surface around the central hole play important roles in DNA binding. The novel ring-shaped architecture of the RdgC dimer has significant implications for its role in homologous recombination.     

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.     

Regulation of bacterial RecA protein function

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.     

Recombination-Dependent Growth in Exonuclease-Depleted Recbc Sbcbc Strains of Escherichia Coli K-12

Analysis of the aroLM-sbcCD interval of the Escherichia coli K-12 chromosome revealed a new gene (rdgC) encoding a function required for growth in recombination-deficient recBC sbcBC strains. Deletion of rdgC does not reduce viability, conjugational recombination, or DNA repair in rec(+), recA, recB, recF, or recJ mutants. However, it makes the growth of recBC sbcBC strains reliant on the RecA, RecF, and RuvC proteins and, to a large extent, on RuvAB. The recBC sbcBC ΔrdgC ruvAB construct forms colonies, but cell viability is reduced to <5%. A recBC sbcBC ΔrdgC derivative carrying the temperature-sensitive recA200 allele grows at 32° but not 42°. Multicopy rdgC(+) plasmids reduce the growth rate of recBC sbcBC strains, while multicopy sbcC(+) plasmids that reactivate SbcCD nuclease cannot be maintained without RdgC protein. The data presented are interpreted to suggest that exonuclease-depleted recBC sbcBC strains have difficulty removing the displaced arm of a collapsed replication fork and that this problem is compounded in the absence of RdgC. Recombination then becomes necessary to repair the fork and allow chromosome duplication to be completed. The possibility that RdgC is an exonuclease is discussed.     

Regulation of Bacterial RecA Protein Function

ABSTRACT

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.     

Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance

The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution. Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A. Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA. Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding.     

Purification and characterization of the single-stranded DNA binding protein from Streptococcus pneumoniae

The Escherichia coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for the RecA protein-promoted three-strand exchange reaction. An open reading frame encoding a protein similar in size and sequence to the E. coli SSB protein has been identified in the Streptococcus pneumoniae genome. The open reading frame has been cloned, an overexpression system has been developed, and the protein has been purified to greater than 99% homogeneity. The purified protein binds to ssDNA in a manner similar to that of the E. coli SSB protein. The protein also stimulates the S. pneumoniae RecA protein and E. coli RecA protein-promoted strand exchange reactions to an extent similar to that observed with the E. coli SSB protein. These results indicate that the protein is the S. pneumoniae analog of the E. coli SSB protein. The availability of highly-purified S. pneumoniae SSB protein will facilitate the study of the molecular mechanisms of RecA protein-mediated transformational recombination in S. pneumoniae.     

Head to head dimer model; an alternative model for the strand exchange reaction by RecA protein of Escherichia coli

The RecA protein of E coli promotes a strand exchange reaction in vitro which appears to be similar to homologous genetic recombination in vivo. A model for the mechanism of strand transfer reaction by RecA protein has been proposed by Howard-Flanders et al based on the assumption that the RecA monomer has two distinctive DNA binding sites both of which can bind to ssDNA as well as dsDNA. Here, I propose an alternative model based on the assumption that RecA monomer has a single domain for binding to a polynucleotide chain with a unique polarity. In addition, the model is based on a few mechanical assumptions that, in the presence of ATP, two RecA molecules form a head to head dimer as the basic binding unit to DNA, and that the binding of RecA protein to a polynucleotide chain induces a structural change of RecA protein that causes a higher state of affinity for another RecA molecule that is expressed as cooperativy. The model explains many of the biochemical capabilities of RecA protein including the polar polymerization of RecA protein on single stranded DNA and polar strand transfer of DNA by the protein as well as the formation of a joint DNA molecule in a paranemic configuration. The model also presents the energetics in the strand transfer reaction.     

AFM studies on the role of the protein RdgC in bacterial DNA recombination

Genetic studies of rdgC in different bacterial systems suggest that it may play a role in replication and recombination. However, the exact function of the corresponding protein, RdgC, is unknown. In this study, we have imaged complexes of RdgC with both linear and supercoiled circular plasmid DNA using atomic force microscopy. We confirm that RdgC does not target any specific sequences in double-stranded DNA, as has been suggested from biochemical data. However, we detect an increased affinity of the protein to DNA ends, and an ability to promote bending of DNA. Similar binding preferences have been reported for enzymes involved in recombination. Protein complexes with supercoiled plasmid DNA further enabled us to study the effect of RdgC on DNA superstructure. At high concentrations of protein we observed promotion of DNA condensation. Recombination is largely enhanced by close contacts of distant regions along the DNA strands, as can occur, for instance, through condensation. Our data thus support a possible function of RdgC as a midwife of recombination.     

Modulating cellular recombination potential through alterations in RecA structure and regulation

The wild-type Escherichia coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to sixfold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to fourfold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50-fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of E. coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function - filament formation and the inherent DNA pairing activity of the formed filaments.     

Biochemical basis of the constitutive coprotease activity of RecA P67W protein

The mutation of Pro67 to Trp (P67W) in the Escherichia coli RecA protein results in reduced recombination and constitutive coprotease phenotypes. We examined the biochemical properties of this mutant in an effort to understand these altered behaviors. We find that RecA P67W protein can access single-stranded DNA (ssDNA) binding sites within regions of secondary structure more effectively than wild-type protein, and binding to duplex DNA is both faster and more extensive as well. This mutant is also more effective than wild-type RecA protein in displacing SSB protein from ssDNA. An enhancement in SSB protein displacement has been shown previously for RecA441, RecA730, and RecA803 proteins, and similarly, this improved ability to displace SSB protein for RecA P67W protein correlates with an increased rate of association with ssDNA. As for the aforementioned mutant RecA proteins, we expect that this enhanced activity will allow RecA P67W protein to bind ssDNA naturally occurring in undamaged cells and to constitutively induce the SOS response. The DNA strand exchange activity of RecA P67W protein is also altered. Although the rate of duplex DNA uptake into joint molecules is increased compared to that of wild-type RecA protein, the resolution to the nicked circular dsDNA product is reduced. We suggest that either a limited amount of DNA strand reinvasion or a defect in DNA heteroduplex extension is responsible for the impaired recombination ability of this mutant protein.     

Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1

The double substitution of Glu156 with Leu and Gly157 with Val in the Escherichia coli RecA protein results in a severely reduced level of recombination and constitutive coprotease behavior. Here we present our examination of the biochemical properties of this mutant protein, RecA N99, in an effort to understand its phenotype and the role of loop 1 (L1) in RecA function. We find that RecA N99 protein has reduced single-stranded DNA (ssDNA)-dependent ATP hydrolysis activity, which is not as sensitive to the presence of SSB protein as wild-type RecA protein. RecA N99 protein is also nearly unable to utilize duplex DNA as a polynucleotide cofactor for ATP hydrolysis, and it shows both a decreased rate of association with ssDNA and a diminished capacity to bind DNA in the secondary binding site. The mutant protein has a corresponding reduction in DNA strand exchange activity, which probably results in the decrease in recombination activity in vivo. The constitutive induction of the SOS response may be a consequence of the impaired ability to repair damaged DNA, resulting in unrepaired ssDNA which can act as a cofactor for the cleavage of LexA repressor. These findings point to an involvement of L1 in both the primary and secondary DNA binding sites of the RecA protein.     

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.     

Effect of RecF protein on reactions catalyzed by RecA protein.

RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli. In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel. The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative. Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins. We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA. It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein. We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA. We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation. The insight that these data give to the normal functioning of RecF protein is discussed.     

Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.     

Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.     

ATP-mediated conformational changes in the RecA filament

The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.     

UvrD controls the access of recombination proteins to blocked replication forks

Blocked replication forks often need to be processed by recombination proteins prior to replication restart. In Escherichia coli, the UvrD repair helicase was recently shown to act at inactivated replication forks, where it counteracts a deleterious action of RecA. Using two mutants affected for different subunits of the polymerase III holoenzyme (Pol IIIh), we show here that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. A defective UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). We propose that UvrD prevents RecA binding, possibly by counteracting RarA. In contrast, at forks affected for the Pol IIIh clamp (DnaN), RarA is not required for RecA binding and the ATPase function of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. UvrD action on RecA is conserved in evolution as it can be performed in E. coli by the UvrD homologue from Bacillus subtilis, PcrA.