©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutations at Proin the RecA Protein P-loop Motif Differentially Modify Coprotease Function and Separate Coprotease from Recombination Activities (*)

Jukka T. Konola (§) , Horacio G. Nastri , Karen M. Logan , Kendall L. Knight

From the (1) Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Prois unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)and (dT), as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro Lys) and homologous recombination (Pro Lys and Pro Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Prois located in a functionally important position in the RecA structure.


INTRODUCTION

The bacterial RecA protein plays at least two distinct roles related to its function as a DNA repair enzyme. RecA catalyzes a strand exchange activity between homologous DNAs (reviewed in Refs. 1-4) and also, in response to DNA damage, becomes activated for a coprotease function (5, 6) . In this latter role RecA mediates the autoproteolysis of the LexA repressor, which in turn increases the expression of a number of genes including recA involved in cell survival following DNA damage (7, 8) . Both the recombination and coprotease functions require a nucleoside triphosphate (NTP) cofactor; however, coprotease activity requires only NTP binding (9, 10) , whereas completion of DNA strand exchange reactions depends on NTP hydrolysis as well (reviewed in Ref. 11). The ATP binding site of the RecA protein contains a consensus phosphate binding loop (P-loop)()motif, a highly conserved element of sequence (12, 13) and structure (14) that plays a central role in coupling NTP binding and hydrolysis events to enzyme activity (13) . The RecA P-loop motif is defined by the sequence GPESSGKTTand is absolutely conserved among all eubacterial RecA proteins for which the sequence is known (1) , suggesting functional and/or structural importance for each residue.

In previous mutagenesis studies, which focused on the recombination activities of RecA, we found that while the identity of the consensus residues in this motif is strictly defined (Gly, Gly, Lys, and Thr), the other residues supported varying levels of substitution (15, 16) . In this study we have compared the effects of mutations in the P-loop on the LexA coprotease and recombination activities of RecA and find that most mutations have similar effects on both functions. However, mutations at Procause differential effects on these activities. One of these mutants, Pro Trp, displays a high constitutive coprotease activity, i.e. activity in the absence of DNA damage. Repressor cleavage assays using the purified mutant protein show that the catalytic properties are similar to other constitutive RecA mutant proteins despite the fact that the amino acid substitutions occur within different areas of the protein structure.

We also describe the first characterization of mutants with a rec/coprtphenotype, i.e. mutants that are completely defective for LexA coprotease activity in vivo but retain some level of recombinational DNA repair and/or homologous recombination activity. Our results are discussed in the context of the crystal structure of the RecA protein (14, 17) and provide information regarding the relationship of certain P-loop residues and mutant side chains to RecA function.


EXPERIMENTAL PROCEDURES

Materials

MacConkey-lactose plates were prepared according to the manufacturer (Difco) and contained 0.5% lactose and 100 µg/ml ampicillin. Other media (LB broth and LB agar) were prepared as described (18) and contained 100 µg/ml ampicillin. Stock solutions (4 mg/ml) of ONPG ( o-nitrophenyl-- D-galactopyranoside; Sigma) were made in Z-buffer (19) . Mitomycin C was from Sigma or Boehringer Mannheim. Protein concentrations in cell extract supernatants were determined using a protein assay kit from Bio-Rad or Pierce. Purified LexA protein was a generous gift from Donald Shepley and John Little (Department of Biochemistry, University of Arizona) and purified cI repressor was a generous gift from Bronwen Brown and Bob Sauer (Department of Biology, Massachusetts Institute of Technology). NTPs, dNTPs, and ddNTPs were from Pharmacia Biotech Inc. Radiolabeled NTPs and dNTPs were from DuPont NEN. Single-stranded RV-1 DNA, an M13 derivative (20) , was used for in vitro repressor cleavage and NTPase assays and was purified as described (21) . Defined length (dA) and (dT) oligonucleotides were made using an Applied Biosystems model 392 DNA/RNA synthesizer.

Strains and Plasmids

All mutant recA genes in this work are carried on plasmids derived from pTRecA103, a pZ150-based plasmid (21) which has the wild type recA gene under control of P(15) . Mutations were introduced using cassette mutagenesis and have been described previously (16) . Plasmid pZ150 (21) was used as a recAcontrol plasmid in the in vivo assays described below. Escherichia coli strain DE1663`, which is recA and carries the lacZ and Y genes under control of the recA operator/promoter, was used for in vivo assays of both DNA repair and LexA coprotease activities of all plasmid borne recA mutants. DE1663` was constructed by mating strain DE1663, a ( recA-srlR) 306::Tn 10 ( lac-argF) U169 sulA211 malB::Tn 9 ( cI ind-1 recAo/p:: lacZY) derivative of AB1157, with strain DE1781 which carries an F` lacIlacPL8 lacZ4505::Tn 5 proABepisome. Both DE1663 and DE1781 were generous gifts from Don Ennis (National Institutes of Health).

Cellular Levels of RecA Protein

All in vivo assays described below were performed in the absence of isopropyl-1-thio-- D-galactopyranoside. Under these conditions the basal level of expression of recA from pTRecA103 is approximately 20-fold greater than from the chromosomal recA gene in wild type E. coli (15) . This increased basal level of expression and the inability of DNA damaging agents to increase recA expression have little or no effect on the ability to distinguish different classes of mutants using assays for both the recombination and coprotease function of RecA (15, 16, 22, 23) . Western blot analysis showed that all mutants in this study produced levels of RecA protein comparable to that from the wild type recA plasmid (15) .

RecA-mediated LexA Cleavage in Vivo

Because strain DE1663` carries the lacZ and Y genes under control of a LexA-regulated promoter measurement of -galactosidase activity is directly related to the extent of RecA-mediated LexA cleavage. Two assays were used for the measurement of -galactosidase activity. 1) Cultures carrying plasmid borne recA mutants were grown overnight in LB-ampicillin medium at 37 °C and diluted 1:40 in the same medium, and 1.5 µl was spotted onto two MacConkey-lactose plates, one containing 0.05 µg/ml mitomycin C and the other containing no mitomycin C. Plates were incubated overnight at 37 °C. Colonies that express -galactosidase activity were red, whereas those in which lacZ remains repressed by LexA were white. Control strains, DE1663`/pTRecA103 ( recAcoprt) and DE1663`/pZ150 ( recAcoprt), were included on each plate. 2) To obtain a more quantitative measure of RecA-mediated LexA cleavage, we determined the specific activity of -galactosidase in the cell extract of each recA mutant. Overnight cultures were grown in LB-ampicillin medium, diluted 1:100 in 6 ml of the same medium, and grown for 2 h at 37 °C. Cultures were divided in half, and to one half mitomycin C was added to a final concentration of 0.5 µg/ml. Incubation was continued for 40 min, cultures were chilled on ice for 20 min, and 1.5 ml of cells were pelleted by centrifugation at 4 °C. Cells were washed in 10 m M NaCl (1 ml) and resuspended in 1 ml Z buffer (19) . Cells were lysed by sonication on ice for 30s, centrifuged for 20 min, and supernatants were stored at 4 °C. -Galactosidase activity in the supernatants was determined using ONPG as described (19) . Units of -galactosidase activity are defined as 10mol ONPG hydrolyzed/min, and activity is expressed as units/µg protein. Extracts from control strains (see above) were prepared fresh along with samples for each experiment. To determine the effect of increased time of exposure to mitomycin C on the specific activity of -galactosidase, samples of control strains and selected mutants were processed at 60, 90, 120, and 180 min as well as 40 min.

Recombination Phenotype of recA Mutants

The ability of all mutants in this study to carry out homologous genetic recombination and recombinational DNA repair has been described (16) . Homologous genetic recombination was measured by determining the plating efficiency of a redgamChi phage on lawns of recA mutants and recombinational DNA repair was measured by observing cell survival following exposure to 4-nitroquinoline oxide and UV light (16) . In the present study, time courses of UV exposure were performed and fractional survival at 30 s relative to the positive control was calculated from the slope of a line obtained from a plot of relative growth versus time of exposure. For certain mutants survival time courses were performed using several different intensities of UV light. UV intensity was measured using a UVX radiometer (UVP, Inc., San Gabriel, CA). We note that recalibration of the radiometer showed that the UV dose in Ref. 16 was 1.52 J/minstead of the 0.67 J/mindicated.

Protein Purification

Both wild type RecA and the Pro Trp mutant RecA proteins were purified using the following procedure. All cultures were grown in 2 LB containing 3.8 mg/ml glucose and 100 µg/ml ampicillin. Four 1.5-liter cultures were inoculated with 50 ml of an overnight culture grown at 37 °C. Incubation at 37 °C was continued until A 0.8, at which time isopropyl-1-thio-- D-galactopyranoside was added to a final concentration of 5 m M. Incubation was continued for 3.5 h, cells were harvested by centrifugation, and pellets resuspended (25 ml/1.5-liter culture) in a buffer containing 0.25 M Tris-HCl, pH 7.5, 25% sucrose. Cell suspensions were quick frozen using liquid nitrogen and stored at -70 °C.

Cell lysis and extraction of the RecA protein using polyethylenimine was performed as described (24) to generate fraction II. Fraction II was dialyzed extensively against R buffer (20 m M Tris-HCl, pH 7.5, 5% glycerol, 5 m M -mercaptoethanol, 0.1 m M EDTA) containing 50 m M NHCl, loaded onto a DE-52 column (30-ml bed volume) equilibrated in the same buffer, and proteins were eluted with a linear gradient (300 ml) of 50-500 m M NHCl. Both wild type RecA and the Pro Trp mutant proteins eluted at approximately 180-280 m M NHCl. Fractions containing RecA protein were pooled and proteins precipitated by addition of ammonium sulfate. The resulting protein pellet was dissolved in 3 ml of R buffer, 30 m M NHCl and dialyzed extensively against the same to generate fraction III. MgClwas then added to a final concentration of 15 m M, and the sample was loaded onto a Sephacryl S-1000 gel filtration column (1.5 120 cm) equilibrated in R buffer, 50 m M NHCl, 15 m M MgCl. Wild type RecA and the Pro Trp mutant proteins elute in pure form in the void volume of this column. Fractions containing RecA were pooled and protein precipitated as above. The precipitate was dissolved in R buffer (200-400 µl) and dialyzed extensively against the same. Glycerol was added to a final concentration of 25%, and samples (20 µl) were quick frozen and stored at -70 °C. RecA proteins were judged to be at least 95% pure in silver-stained SDS-polyacrylamide gels. The concentration of wild type RecA protein was determined spectrophotometrically using an extinction coefficient of = 0.59 mgml (25) . The concentration of the Pro Trp mutant protein was determined using the Bio-Rad protein assay kit, the BCA protein assay kit from Pierce and by comparing the Coomassie staining intensity of a number of different samples of mutant with known amounts of wild type RecA protein on SDS-polyacrylamide gels. These determinations corresponded to an extinction coefficient for the Pro Trp mutant of = 1.2 mgml. The same was recently calculated for a His Trp mutant RecA protein (26) .

RecA-mediated Repressor Cleavage in Vitro

The cleavage of LexA repressor by purified RecA protein (wild type and the Pro Trp mutant) was determined using the buffer described by Wang et al. (27) . Reaction mixtures (40 µl) contained 1 µ M wild type or mutant RecA protein, 1.0 m M of the indicated NTP cofactor, and 35 µ M RV-1 single-stranded DNA or the indicated oligonucleotide cofactor (concentration of DNA expressed as mol PO). Alternative nucleic acid cofactors were used as indicated. Reactions were started with the addition of LexA protein to a final concentration of 6 µ M. Samples (8 µl) were removed at the indicated times, added to gel loading buffer (10 µl), heated at 95 °C for 3 min, and loaded onto 15% polyacrylamide gels containing SDS. Gels were stained with Coomassie Brilliant Blue R, and the percentage of LexA cleavage was determined by scanning densitometry of intact LexA and LexA cleavage products (Biomed Instruments 1D/2D soft laser scanning densitometer). Cleavage of cI repressor was performed essentially as described (28) . Reaction mixtures (10 µl) contained 10 µ M wild type or mutant RecA, 35 µ M of the indicated oligonucleotide cofactor, 0.5 m M ATPS, and 1 µ M cI repressor. Following a 3.5-h incubation at 37 °C the reaction was analyzed on SDS-polyacrylamide gels as described for LexA cleavage.

RecA Protein NTPase Activity

Hydrolysis of -P-labeled ATP, dATP, GTP, dGTP, CTP, dCTP, TTP, and UTP by purified RecA (wild type and Pro Trp mutant) was measured essentially as described (29) . Reactions included the following components: 20 m M Tris-HCl (pH 7.5), 20 m M KCl, 10 m M MgCl, 0.5 m M EDTA, 1.0 m M dithiothreitol, 0.5 m M -P labeled NTP (20 µCi/ml), 2.0 µ M RecA protein (wild type or Pro Trp mutant), and 25 µ M RV-1 single-stranded DNA. Percent NTP hydrolysis was measured by scanning polyethylenimine chromotography plates using a Molecular Dynamics PhosphorImager equipped with Imagequant software (version 5.6).


RESULTS

We have previously characterized 100 recA mutants carrying single amino acid substitutions in the P-loop (residues 66-74) regarding their ability to perform recombinational DNA repair and homologous genetic recombination in vivo (16) . Thirty-seven of these mutants were fully or partially active for one or both of these functions, and the remaining 63 were completely inhibited for both (16) . In this study we have measured both the coprotease and DNA repair activities and compared the effects of each mutation on these different functions. We used two in vivo assays to measure the RecA coprotease activity toward LexA repressor and have been able to separate all mutants into three phenotypic categories: coprt(constitutive), coprt(inducible), and coprt. Mutants are defined as coprtif they form red colonies on MacConkey-lactose plates in the absence of mitomycin C (non-inducing condition) and if the average non-induced -galactosidase activity in the cell extract is at least 1.5-fold greater than that of the positive control strain (DE1663`/pTRecA103). Mutants are defined as coprtif they form white colonies on MacConkey-lactose plates in both the absence and presence of mitomycin C and have -galactosidase activity in the cell extract comparable to that of the negative control strain (DE1663`/pZ150). Mutants are defined as coprtif they form white colonies when grown on MacConkey-lactose plates in the absence of mitomycin C, form red colonies when grown in the presence of mitomycin C, and show mitomycin C induction of -galactosidase activity in the cell extract.

coprtMutants

Six of the 37 recombination-proficient mutants showed constitutive coprotease activity, and in each case the amino acid substitution occurs at either Proor Thr(Table I). The Pro Trp mutant (, 67-10) is unique in that it has high coprotease activity in the absence of mitomycin C, whereas substitution of Prowith Glu, Asp, or Gly and Thrwith Gly or Phe results in low level coprtmutants. In an extended time course of exposure to mitomycin C, coprtmutants with substitutions at Pro(Trp, Glu, and Asp) show an increase in coprotease specific activity similar to wild type RecA (Fig. 1, A and B). Because the expression of recA is under the control of Pand, therefore, is unaffected by exposure to DNA damaging agents, this increase in specific activity may reflect a time-dependent activation and/or a slower rate of degradation of the existing pool of RecA as DNA damage accumulates.

These coprtmutations show varying effects on recombinational DNA repair activity. For example, although the enhanced effect on coprotease activity is similar for both a Glu and Asp substitution at Pro(), the Glu mutation has a greater inhibitory effect on recombinational DNA repair (Table II and Ref. 16). In addition, only the Pro Trp mutant results in a high constitutive coprotease activity, but substitution to Trp and Gly at position 67 and to Gly and Phe at position 74 all have similar modest inhibitory effects or no measurable effect on recombinational DNA repair ( and Ref. 16).

coprtMutants

Twenty-nine of the 37 recombination proficient mutants scored as coprt, and although most mutants showed levels of induction comparable to wild type RecA, three showed low levels of inducible coprotease activity; Pro Phe, Pro Tyr, and Glu Thr (; 67-11, 67-12, and 68-6, respectively). The Pro Phe mutant is most attenuated for coprotease induction and required longer times of exposure to mitomycin C before showing a measurable increase in activity (Fig. 1, C and D). Inducible coprotease activity was clearly observed for all coprtmutants, including Pro Phe, on MacConkey-lactose plates showing that this method is also suitable for detection of low levels of coprotease induction. coprtMutants-Two mutants previously characterized as having partial recombination activity, Pro Lys and Pro Arg, showed a complete lack of coprotease activity in vivo (, 67-14 and 67-16). Both mutants grew as white colonies on MacConkey-lactose plates in the absence and presence of mitomycin C and showed a level of coprotease activity in cell extracts similar to negative control cells (). Even after prolonged exposure to mitomycin C, neither mutant showed induction of coprotease activity (Fig. 1, C and D). We have further tested the DNA repair proficiency of these two mutants using a range of UV doses (). We find that the Pro Lys mutant shows a steady increase in survival, up to 33% of wild type recA cells, with decreasing UV dose. In contrast, the Pro Arg mutant fails to survive even low doses to any extent greater than negative control cells. Interestingly, both mutants are proficient for RecA-dependent homologous recombination as they promote the formation of plaques by a redgamChi phage (16) . To our knowledge this is the first description of recA mutants that are proficient for recombinational DNA repair and/or homologous genetic recombination but are completely deficient for LexA coprotease activity in vivo.


Figure 1: RecA coprotease activity as a function of time of cell growth and exposure to mitomycin C. -galactosidase activity in crude cell extracts was determined as described under ``Experimental Procedures'' at 40, 60, 90, 120, and 180 min of cell growth in the absence ( A and C) or presence ( B and D) of 0.5 µg/ml mitomycin C. 67Trp, 67Glu, and 67Asp ( A and B) are examples of coprtmutants. 67Phe ( C and D) shows a low level induction of activity, whereas 67Lys and 67Arg ( C and D) are examples of coprtmutants that show no induction of activity. The positive control is strain DE1663` carrying pTRecA103 and the negative control is the same strain carrying pZ150 (see ``Experimental Procedures''). Values are an average of duplicate assays for which the standard error was no greater than 18%.



All 63 of the P-loop mutants that have been previously characterized as defective for both recombinational repair and homologous recombination (16) scored as coprt(data not shown; Fig. 2).

A summary of all mutations and their effect on coprotease activity is shown in Fig. 2.


Figure 2: Summary of mutations and their effect on RecA coprotease activity. The wild type RecA sequence is centered in boldface type. Substitutions shown above allow coprotease activity and are listed from top to bottom in decreasing order of the level of activity seen in the absence of mitomycin C (see Table I). Underlines indicate constitutive coprotease activity. Substitutions shown below the wild type sequence result in coprtmutants. This summary is derived exclusively from single mutants.



Nucleoside Triphosphate Cofactor Specificity for LexA Cleavage by the Pro Trp Mutant

In order to investigate the molecular nature of the high constitutive coprotease activity of the Pro Trp mutation, we have begun biochemical studies of the purified mutant protein. Studies of other mutant RecA proteins have shown that constitutive activation of coprotease function can be associated with a more relaxed specificity for NTP cofactors (27) . We, therefore, tested all of the common NTPs, dNTPs, and ddNTPs in an in vitro LexA cleavage assay and found that the Pro Trp mutant protein shows a completely relaxed specificity compared to wild type RecA. Under the conditions of our assay ATP, dATP, and ddATP were the optimal cofactors for both wild type RecA and the Pro Trp mutant protein (Fig. 3 A). In the presence of any one of these three cofactors the initial rate of LexA cleavage was quite fast, ranging from 60 to 78% repressor cleavage after 5 min. Both proteins also utilized CTP and UTP equally well, although the initial rate and final extent of LexA cleavage was somewhat less than with ATP, dATP, and ddATP (Fig. 3, B and D). However, the mutant protein was able to use both dCTP and ddCTP more effectively than wild type RecA (Fig. 3 B). In sharp contrast to the above NTPs, we found that the Pro Trp mutant protein was able to catalyze LexA cleavage in the presence of GTP, dGTP, ddGTP, TTP, and ddTTP, whereas wild type RecA was completely ineffective with these nucleotides (Fig. 3, C and D). Neither wild type RecA nor the Pro Trp mutant protein showed any cleavage of LexA when ADP was used as cofactor or when ssDNA or NTPs were omitted from the reaction mixture (data not shown).


Figure 3: Nucleotide cofactor specificity for LexA cleavage activity by both wild type RecA and the Pro Trp mutant proteins. LexA cleavage assays were performed as described under ``Experimental Procedures.'' Each panel shows percentage of LexA cleavage as a function of time for both the wild type and mutant proteins. NTP cofactors and symbols are identified in each panel. Values are the average of duplicate assays for which the standard error was no greater than 15%.



Despite the fact that the nucleotide specificity for LexA cleavage is relaxed by the Pro Trp mutation, NTP hydrolysis by this mutant still shows a pattern of specificity similar to wild type RecA. We determined specific activities for the hydrolysis of several NTPs and dNTPs. Both wild type and mutant RecA proteins hydrolyzed dATP and ATP most efficiently (Table III). Hydrolysis of other nucleotides (UTP, CTP, dCTP, GTP, dGTP, and TTP) by both proteins was significantly less efficient, with GTP and TTP being the least effective substrates. These results are in good agreement with those of Weinstock et al. (29) regarding the nucleotide specificity for wild type RecA NTPase activity. Hydrolysis of NTPs by either protein required single-stranded DNA as a cofactor.

Nucleic Acid Cofactor Specificity for Repressor Cleavage by the Pro Trp Mutant

Previous studies have shown that in addition to a more relaxed specificity for NTP cofactors, coprtmutant RecA proteins may also have a relaxed specificity for nucleic acid cofactors (28, 30) . McEntee and Weinstock (28) have shown that the coprtRecA441 protein can use shorter length (dA) and (dT) oligonucleotides than can wild type RecA as cofactors for the cleavage of the cI repressor. However, no study has yet reported the effect of defined length oligonucleotides on the LexA cleavage activity of either wild type or any mutant RecA proteins. We, therefore, compared the effectiveness of a series of short (dA) and (dT) oligonucleotides as cofactors in LexA cleavage assays using both wild type RecA and the Pro Trp mutant protein. Interestingly, our data show that the LexA coprotease activity of both proteins is only minimally affected by changes in the length of the oligonucleotide cofactor in the range from 6 to 24 bases (Fig. 4). For example, the extent of LexA cleavage by wild type RecA in the presence of (dA)or (dA)is 30% and 37%, respectively. For the mutant protein the difference in these values is somewhat greater, 35% versus 51%, respectively. Using (dT)or (dT), the extent of LexA cleavage by wild type RecA is 48% and 60%, while cleavage by the Pro Trp protein is 57% and 71%, respectively. With all lengths of (dA) and (dT) oligonucleotides, the mutant protein catalyzes LexA cleavage to an extent approximately 15-30% greater than wild type RecA. We also performed time courses of LexA cleavage in which the concentration of oligonucleotides was varied from 8 µ M to 32 µ M with protein held constant at 1 µ M. In all cases both the initial rate and extent of cleavage increased with increasing concentration of (dA) or (dT) cofactor, and again the activity of the mutant protein was approximately 15-30% greater than wild type RecA (data not shown). Also apparent in Fig. 4is that (dT) oligonucleotides are slightly more effective cofactors than the corresponding (dA) oligonucleotide.


Figure 4: The effect of nucleic acid cofactor length on the cleavage of LexA repressor by wild type RecA and the Pro Trp mutant proteins. Percentage of LexA cleavage was determined following a 20-min reaction containing 1 m M dATP and 35 µ M oligonucleotide cofactor (dA, panel A; dT, panel B) of the indicated length. Other reactions conditions and components are described under ``Experimental Procedures.'' Values are the average of triplicate assays, and the standard error is indicated in the figure.



Because our data regarding LexA cleavage by wild type RecA differed significantly from work that showed a strict dependence on oligonucleotide length for cI repressor cleavage by wild type RecA (28) , we used our set of (dA) and (dT) oligonucleotides in repressor cleavage assays. We found that, in contrast to LexA, cleavage of repressor does indeed show a marked dependence on oligoucleotide length (Fig. 5). Using (dA) oligonucleotides, we observed a steady increase in the extent of repressor cleavage by both wild type RecA (from 0% to 70% cleavage) and the Pro Trp protein (from 17% to 70% cleavage) as a function of increasing length of oligonucleotide (Fig. 5 A). With (dA)and (dA), the mutant protein showed a reproducibly higher activity than wild type RecA, but both proteins had essentially identical activities with (dA), (dA), and (dA). Using (dT), however, we observed significant differences between the wild type and mutant proteins (Fig. 5 B). With (dT), (dT)and (dT), wild type RecA was essentially inactive, whereas the mutant protein displayed measurable activity with (dT)and (dT), which increased dramatically with (dT). A significant level of activity was seen for wild type RecA using (dT)and was approximately 50% the level observed for the mutant protein. Activity was equivalent for both proteins using (dT). Although somewhat different in their quantitative aspects, these results are consistent with those reported previously for wild type RecA (28) .


Figure 5: The effect of nucleic acid cofactor length on the cleavage of cI repressor by wild type RecA and the Pro Trp mutant proteins. Percentage of cI cleavage was determined as described under ``Experimental Procedures.'' Oligonucleotide cofactor was either dA or dT of the indicated length. Values are the average of triplicate assays, and the standard error is indicated in the figure.



Wang et al. (30) have reported that the coprtrecA1202 and recA1211 mutant proteins can use rRNA and tRNA very effectively as cofactors for cleavage of LexA and also showed that these mutant proteins use double-stranded DNA somewhat more effectively than does wild type RecA. We tested the ability of the Pro Trp mutant protein to use these alternative nucleic acid cofactors and found that tRNA and double-stranded DNA are unable to function in this capacity (data not shown).


DISCUSSION

In the present study we have analyzed 100 unique recA mutants, each carrying a single amino acid substitution in one of 9 residues that define the P-loop motif, and we find that Prois unique among them in that specific substitutions have very different effects on the coprotease versus recombination activities of RecA. One of these, Pro Trp, results in high constitutive coprotease activity and a modest effect on recombinational DNA repair activity, whereas coprtmutants resulting from Glu and Asp substitutions markedly decrease the latter activity. In contrast, Lys and Arg mutations at position 67 eliminate in vivo coprotease function but allow for some level of recombination activity. The Lys mutant catalyzes homologous genetic recombination (16) and maintains a modest level of recombinational DNA repair activity, whereas the Arg mutant, while capable of carrying out homologous recombination (16) , is completely inactivated for recombinational DNA repair. These two represent the first description of mutants that separate the coprotease, recombinational DNA repair and homologous recombination functions of RecA. The identification of such mutants undoubtedly relates to the fact that expression of recA in our system is regulated by a non-SOS promoter, P. Under normal SOS regulation the in vivo recombination activity of coprtmutants is likely to be refractory to analysis simply because recA expression is repressed.

We have used the RecA/ADP crystal structure (14, 17) as a model for the assessment of the data presented here. In this structure the main chain atoms of the RecA P-loop motif are arranged in a remarkably similar orientation to those in the P-loop motif of adenylate kinase, elongation factor Tu (EF-Tu), and p21 (14) , suggesting a conservation of the functional properties of these residues. For RecA, EF-Tu, and p21, the - and -phosphates of bound nucleotide are positioned similarly but the location of the base and sugar in the RecA-ADP complex is very different from that for the GDP of EF-Tu and p21 (14) . Electron microscopy studies suggest that the RecA crystal structure represents a specific inactive conformation (reviewed in Ref. 31); however, the overall structure of the crystal filament closely resembles the active RecA/DNA/ATP filament (31) .

The crystal structure of RecA shows that the ATP binding site is near the surface of each protein monomer such that it lies on the inner side of the helical, oligomeric filament (Fig. 6; also see Refs. 14, 16, and 17). The side chains of 3 P-loop residues (Pro, Glu, and Ser) extend inward toward the helical axis of the protein filament and appear to be free of any steric impedance. However, each of these positions, especially 68 and 69, shows restrictions regarding mutations that permit recombination activity (16) . We have previously suggested that these restrictions reflect a constraint that is not apparent in the crystal structure, namely their interaction with, or at least proximity to, bound DNA (16) . This is supported by recent work in which a specific cross-link between poly(dT) and RecA was identified within a peptide defined by residues Ileto Lys.()

We have found that for positions 68 and 69 all mutations have equivalent effects on the coprotease and recombination activities. Because DNA is a required cofactor for the coprotease activity, mutations that alter DNA binding may effect both functions similarly. However, at position 67 mutations occur that differentially modify the recombination and coprotease activities. A particularly striking example of this is seen by comparing the Lys and Glu mutants, two substitutions that have similar inhibitory effects on the recombination activities but precisely opposite effects on the coprotease activity. Pro Glu results in a low level coprtmutant, while Pro Lys completely inhibits this function. These results are consistent with the idea that position 67 is close to bound LexA repressor and that mutations here may have a direct effect on the interaction between RecA and LexA. This idea is supported by electron microscopic studies of RecA/LexA/DNA complexes in which LexA is seen to bind within the deep helical groove of the RecA filament (32) . Although the wild type side chain of Prodoes not extend far from the inner surface of the protein filament, mutant side chains such as Trp, Lys, and Glu may protrude far enough into the helical groove to have a direct effect on LexA binding (Fig. 6). Another mutant observed at position 67, however, suggests a more indirect effect on RecA function. A Pro Gly mutation results in a modest constitutive coprotease activity, yet has little or no effect on recombination, suggesting that activation of the coprotease function results from increased main chain flexibility in this region of the protein. Whether these mutations correlate with changes in LexA binding affinities will be addressed in biochemical studies of the purified mutant proteins.

Mutations at residues 229 (Gly Ser) and 243 (Arg Leu) differentially affect cleavage of the LexA, cI, and 80 repressors (33, 34) , and both the RecA crystal structure and electron microscopic analysis of RecA/LexA/DNA complexes support the idea that these residues occupy space within the repressor binding site (14, 32) . The distance between residues 67, 229, and 243 in the RecA crystal structure (14 Å separation) allows for the possibility that all could be close to bound repressor (Fig. 6).


Figure 6: Stereo diagram of two neighboring RecA subunits showing the position of Pro and bound ADP. An -carbon trace is shown for two neighboring monomers in the RecA protein filament as seen in the crystal structure (17). The thickness of the polypeptide chain is slightly greater for the right subunit. The side chain and backbone atoms of Proand bound ADP are shown in boldface. The N-terminal residue (Asp) seen in the crystal structure is indicated by N in the left subunit. 229 and 243 refer to Glyand Argand lie immediately to the left of the side chain for each residue in both subunits. In the left subunit, residues that flank the disordered regions L1 and L2 are indicated by a 1 (Glu) and 1` (Gly) and a 2 (Ile) and 2` (Thr). In the right monomer, these 4 residues are shown as a closed circle. This view shows that the Proside chain extends from the inner surface of the protein inward toward the helical axis.



Position 67 is also very near the disordered loop 2 (L2, residues 196-209). Story et al. (17) have proposed that L2 forms part of the primary DNA binding site of RecA and that specific conformational changes in this region that affect DNA binding are regulated by the interaction of Glnwith the -phosphate of bound NTP. Distance measurements within the RecA structure show that the -carbon of any mutant side chain at position 67 would be within 5.0 Å of the Glnside chain and approximately 3.5-13.0 Å from the proximal and distal residues that flank L2, Ileand Thr, respectively (Fig. 6). Therefore, the proximity of substitutions at position 67 to Glnor residues within L2 may contribute to their effects on the coprotease or recombination activities of RecA.

Biochemical studies of the purified Pro Trp mutant protein show a complete relaxation of NTP cofactor specificity for RecA-mediated LexA cleavage in vitro. Although an NTP cofactor is still required for this activity, any ribo-, deoxyribo-, or dideoxyriboNTP that we tested served in this capacity for the mutant protein, whereas wild type RecA displayed a significantly more stringent NTP specificity. Wang et al. (27) have previously reported that two different coprtmutant RecA proteins, recA1202 (Gln Lys) and recA1211 (Glu Lys), display a somewhat relaxed specificity for NTP cofactors in LexA cleavage assays. These findings show that the nucleotide cofactor specificity of coprtmutants can be modified by mutations at very different locations within the RecA structure.

The relaxed nucleotide specificity of the Pro Trp mutant protein pertains to the coprotease activity but not to the NTPase activity. Nucleotide cofactors serve as allosteric effectors of RecA activity (reviewed in Ref. 11), and NTP binding and hydrolysis has been shown to induce specific changes in the structure of the protein (35, 36, 37) . It may be that coprtmutations at position 67 facilitate NTP-induced structural changes required for coprotease function that can now be achieved by otherwise non-optimal NTP cofactors.

We