Suppression of Escherichia coli recF mutations by recA-linked srfA mutations.

Suppressors of recF (srfA) were found by selection for resistance to mitomycin C and UV irradiation in a recB21 recC22 sbcB15 recF143 strain. srfA mutations map in recA and are dominant to srfA+. They suppress both the DNA repair and the recombination deficiencies due to recF mutations. Therefore, RecA protein which is altered by the srfA mutation can allow genetic recombination to proceed in the absence of recB, recC, and recF functions. recF is also required for induction of the SOS response after UV damage. We propose that recF+ normally functions to allow the expression of two recA activities, one that is required for the RecF pathway of recombination and another that is required for SOS induction. The two RecA activities are different and are separable by mutation since srfA mutations permit recombination to proceed but have not caused a dramatic increase in SOS induction in recF mutants. According to this hypothesis, one role for recF in DNA repair and recombination is to modulate RecA activities to allow RecA to participate in these recF-dependent processes.     

Probing the DNA sequence specificity of Escherichia coli RECA protein

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5′-GCTGGTGG-3′). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a λ repressor fragment in the presence of RecA, ADP-AlF₄ and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.     

Use of recA803, a partial suppressor of recF, to analyze the effects of the mutant Ssb (single-stranded DNA-binding) proteins in vivo and in vitro

Summary We examined the possibility that the ssb-1 and ssb-113 mutants exert some of their effects by interfering with the normal function of wild-type RecF protein. Consistent with this possibility, we found that recA803, which partially suppresses recF mutations, also partially suppresses both ssb mutations, as detected by an increase in UV resistance. No evidence was obtained for suppression of the defect in lexA regulon inducibility caused by the ssb mutations. Consequently we suggest that suppression occurs by increasing recombinational repair. In vitro tests of Ssb mutant and wild-type proteins revealed that the single-stranded DNA dependent ATPase activity of RecA protein is more susceptible to inhibition than the joint-molecule-forming activity. All three Ssb proteins inhibit the ATPase activity of RecA wild-type protein almost completely while under similar conditions they inhibit the joint-molecule-forming activity only slightly. Both activities of RecA803 protein were found to be less inhibited by the three Ssb proteins than those of RecA wild-type protein. This is consistent with the suppressing ability of recA803. We found no evidence to contradict the previously proposed hypothesis that ssb-1 affects recombinational repair by acting as a weaker form of Ssb protein. We found, however, only very weak evidence that Ssb-113 protein interferes directly with recombinational repair so that the possibility that it interferes with a normal function of RecF protein must remain open.     

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.     

Problems and Paradigms: Relating biochemistry to biology: How the recombinational repair function of RecA protein is manifested in its molecular properties

The multiple activities of the RecA protein in DNA metabolism have inspired over a decade of research in dozens of laboratories around the world. This effort has nevertheless failed to yield an understanding of the mechanism of several RecA protein-mediated processes, the DNA strand exchange reactions prominent among them. The major factors impeding progress are the invalid constraints placed upon the problem by attempting to understand RecA protein-mediated DNA strand exchange within the context of an inappropriate biological paradigm – namely, homologous genetic recombination as a mechanism for generating genetic diversity. In this essay I summarize genetic and biochemical data demonstrating that RecA protein evolved as the central component of a recombinational DNA repair system, with the generation of genetic diversity being a sometimes useful byproduct, and review the major in vitro activities of RecA protein from a repair perspective. While models proposed for both recombination and recombinational repair often make use of DNA strand cleavage and transfer steps that appear to be quite similar, the molecular and thermodynamic requirements of the two processes are very different. The recombinational repair function provides a much more logical and informative framework for thinking about the biochemical properties of RecA and the strand exchange reactions it facilitates.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.     

Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k(cat)/K(m)) of the hRAD51 ATPase was approximately 50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be approximately 3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.     

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.     

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.     

A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the α-helix H of the C-terminal sub-domain of the protein. recA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42°C) in the absence of ATP resulted in inhibition at 42°C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA–RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5M) was shown to be another factor that can stabilize the RecAmut protein at 42°C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecAmut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.     

Homologous pairing of single-stranded DNA and superhelical double-stranded DNA catalyzed by RecO protein from Escherichia coli.

The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells. RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules. In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein. The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent. Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study. The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival. These results suggest that RecO and RecA proteins may have common functional properties.     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264→Glu) and recA665 (Tyr264→His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

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.     

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60–70°C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.     

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.     

Biochemical events essential to the recombination activity of Escherichia coli RecA protein. II. Co-dominant effects of RecA142 protein on wild-type RecA protein function

In the accompanying paper, RecA142 protein was found to be completely defective in DNA heteroduplex formation. Here, we show that RecA142 protein not only is defective in this activity but also is inhibitory for certain activities of wild-type RecA protein. Under appropriate conditions, RecA142 protein substantially inhibits the DNA strand exchange reaction catalyzed by wild-type RecA protein; at equimolar concentrations of each protein, formation of full-length gapped duplex DNA product molecules is less than 7% of the amount produced by wild-type protein alone. Inhibition by RecA142 protein is also evident in S1 nuclease assays of DNA heteroduplex formation, although the extent of inhibition is less than is observed for the complete DNA strand exchange process; at equimolar concentrations of wild-type and mutant proteins, the extent of DNA heteroduplex formation is 36% of the wild-type protein level. This difference implies that RecA142 protein prevents, at minimum, the branch migration normally observed during DNA strand exchange. RecA142 protein does not inhibit either the single-strand (ss) DNA-dependent ATPase activity or the coaggregation activities of wild-type RecA protein. This suggests that these reactions are not responsible for the inhibition of wild-type protein DNA strand exchange activity by RecA142 protein. However, under conditions where RecA142 protein inhibits DNA strand exchange activity, RecA142 protein renders the M13 ssDNA-dependent ATPase activity of wild-type protein sensitive to inhibition by single-strand DNA-binding protein, and it inhibits the double-strand DNA-dependent ATPase activity of wild-type RecA protein. These results imply that these two activities are important components of the overall DNA strand exchange process. These experiments also demonstrate the applicability of using defective mutant RecA proteins as specific codominant inhibitors of wild-type protein activities in vitro and should be of general utility for mechanistic analysis of RecA protein function both in vitro and in vivo.     

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.