Analysis of double stranded DNA-dependent activities of Deinococcus radiodurans RecA protein

In this study, the double-stranded DNA-dependent activities of Deinococcus radiodurans RecA protein (Dr RecA) were characterized. The interactions of the Dr RecA protein with double-stranded DNA were determined, especially dsDNA-dependent ATP hydrolysis by the Dr RecA protein and the DNA strand exchange reaction, in which multiple branch points exist on a single RecA protein-DNA complex. A nucleotide cofactor (ATP or dATP ) was required for the Dr RecA protein binding to duplex DNA. In the presence of dATP, the nucleation step in the binding process occurred more rapidly than in the presence of ATP. Salts inhibited the binding of the Dr RecA protein to double-stranded DNA. Double-stranded DNA-dependent ATPase activities showed a different sensitivity to anion species. Glutamate had only a minimal effect on the double-stranded DNA-dependent ATPase activities, up to a concentration of 0.7 M. In the competition experiment for Dr RecA protein binding, the Dr RecA protein manifested a higher affinity to double-stranded DNA than was observed for single-stranded DNA.     

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.     

Activation of protease-constitutive recA proteins of Escherichia coli by rRNA and tRNA.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.     

Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates.

To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E. S. Tessman and P. Peterson, J. Bacteriol. 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro. In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition. We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor. The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP. These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein. With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo. The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition.     

Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis

The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.     

Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation

The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.     

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg²⁺-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors.     

Purification and characterization of the RecA protein from Streptococcus pneumoniae

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.     

Bacillus subtilis SsbA and dATP regulate RecA nucleation onto single-stranded DNA

Bacillus subtilis RecA preferentially hydrolyzes dATP over ATP and supports an efficient DNA strand exchange reaction in the presence of dATP when compared to ATP. Saturating amounts of SsbA, independently of the order of addition, reduce the single-stranded (ss) DNA-dependent dATPase activity of RecA, and block the ATPase activity. SsbA added prior to RecA slightly stimulates the dATP-dependent DNA strand exchange activity, whereas added after RecA greatly enhances the extent of strand exchange. In the presence of ATP, 10 times more RecA is required to achieve a comparable level of strand exchange than in the presence of dATP. We propose that dATP binding and hydrolysis as well as SsbA provide different levels of regulation of the dynamic RecA nucleoprotein filament.     

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.     

ADP-dependent DNA strand exchange by the mutant [P67G/E68A] RecA protein

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent three-strand exchange reaction between a circular bacteriophage phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution. These results indicate that the strand exchange activity of the [P67G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted strand exchange reaction that is dependent on ADP. It is likely that the [P67G/E68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type RecA protein.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.     

Characterization of DNA strand transfer promoted by Mycobacterium smegmatis RecA reveals functional diversity with Mycobacterium tuberculosis RecA

The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria.     

Defining the salt effect on human RAD51 activities

Previous work by Sung and colleagues identified unusual salt requirements for hRAD51 strand exchange compared to RecA [S. Sigurdsson, K. Trujillo, B. Song, S. Stratton, P. Sung, Basis for avid homologous DNA strand exchange by human Rad51 and RPA, J. Biol. Chem. 276 (2001) 8798-8806]. Later studies showed that this salt [(NH4)2SO4] appeared to enhance the ability of hRAD51 to distinguish ssDNA from dsDNA [Y. Liu, A.Z. Stasiak, J.Y. Masson, M.J. McIlwraith, A. Stasiak, S.C. West, Conformational changes modulate the activity of human RAD51 protein, J. Mol. Biol. 337 (2004) 817-827]. The mechanism of this salt effect remains enigmatic. Here, we detail the properties of several neutral salts on hRAD51 activities. We found that the cation identity correlated with the stimulatory effect of these neutral salts on hRAD51 ATPase and strand exchange activities. The salt effect appears to be related to the size of the cation, which may be largely mimicked with the cesium ion. These results are consistent with the hypothesis that stimulating cations induce an important conformation and/or transition state in hRAD51. In the presence of an optimal ammonium-based salt (NaNH4HPO4), hRAD51 mediated strand exchange was successfully performed using a simplified protocol. We confirmed and extend the observation that efficient strand exchange correlated with preferential binding of ssDNA over dsDNA. In addition we observed an induced stability of the hRAD51-DNA complex in the presence of ATP that becomes unstable following ATP hydrolysis (the ADP form or nucleotide free form). These salt-induced characteristics of hRAD51 increasingly resemble RecA-mediated recombinase activities, which should help in dissecting the mechanism of these proteins in homologous recombination.     

Three-strand exchange by the Escherichia coli RecA protein using ITP as a nucleotide cofactor: mechanistic parallels with the ATP-dependent reaction of the RecA protein from Streptococcus pneumoniae

The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nucleoside triphosphate, ITP, as the nucleotide cofactor. However, although both reactions are stimulated by single-stranded DNA-binding (SSB) protein, the ITP-dependent reaction differs from the ATP-dependent reaction in that it is observed only at low SSB protein concentrations, whereas the ATP-dependent reaction proceeds efficiently even at high SSB protein concentrations. Moreover, the circular ssDNA-dependent ITP hydrolysis activity of the RecA protein is strongly inhibited by SSB protein (suggesting that SSB protein displaces RecA protein from ssDNA when ITP is present), whereas the ATP hydrolysis activity is uninhibited even at high SSB protein concentrations (because RecA protein is resistant to displacement by SSB protein when ATP is present). These results suggest that SSB protein does not stimulate the ITP-dependent strand exchange reaction presynaptically (by facilitating the binding of RecA protein to the circular ssDNA substrate) but may act postsynaptically (by binding to the displaced strand that is generated when the circular ssDNA invades the linear dsDNA substrate). Interestingly, the mechanistic characteristics of the ITP-dependent strand exchange reaction of the E. coli RecA protein are similar to those of the ATP-dependent strand exchange reaction of the RecA protein from Streptococcus pneumoniae. These findings are discussed in terms of the relationship between the dynamic state of the RecA-ssDNA filament and the mechanism of the SSB protein-stimulated three-strand exchange reaction.     

Purification of a RecA protein analogue from Bacillus subtilis

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.     

Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.     

RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination.     

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.     

Roles of RecA protease and recombinase activities of Escherichia coli in spontaneous and UV-induced mutagenesis and in Weigle repair.

The RecA protein has a second, direct role in the mutagenesis of Escherichia coli and bacteriophage lambda in addition to its first, indirect role of inducing the SOS system by enhancing the proteolytic cleavage of the LexA repressor protein. The need for RecA protease and recombinase functions in the direct role was examined in cells containing split-phenotype RecA mutations, in the absence of LexA protein. Spontaneous mutation of E. coli (his----his+) required both the protease and recombinase activities. The mutation frequency increased with increasing RecA protease strength. In contrast, UV-induced mutation of E. coli required only the RecA protease activity. Weigle repair and mutation of UV-irradiated phage S13 required only RecA protease activity, and even weak activity was highly effective; RecA recombinase activity was not required. RecA+ protein inhibited RecA (Prtc [protease constitutive] Rec+) protein in effecting spontaneous mutation of E. coli. We discuss the nature of the direct role of the RecA protein in spontaneous mutation and in repair and mutagenesis of UV-damaged DNA and also the implications of our results for the theory that SOS-mutable cryptic lesions might be responsible for the enhanced spontaneous mutation in Prtc Rec+ strains.