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Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair1
Article No. jmbi.1998.2515 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 286, 1097±1106
Saturation Mutagenesis of the E. coli RecA Loop L2 Homologous DNA Pairing Region Reveals Residues Essential for Recombination and Recombinational Repair Konstanze HoÈrtnagel, Oleg N. Voloshin, Hai H. Kinal, Ning Ma Carianne Schaffer-Judge and R. Daniel Camerini-Otero* Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda MD 20892-1810, USA
*Corresponding author
The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational pro®ciency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identi®ed as stringently required for recombinational events in bacterial cells. In addition, our ®ndings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming ®lamentous b-structures, we propose how the information from the mutant analysis might facilitate the use of a simpli®ed amino acid alphabet to design b-structure forming L2 peptides with improved RecAlike activities. Keywords: RecA protein; homologous recombination; recombinational repair; DNA pairing; ATPase activity
Introduction The prokaryotic RecA protein is an essential component of many cellular processes, including general homologous recombination, recombinational DNA repair, and induction of the SOS response to DNA damage (Dunderdale & West, 1994; Roca & Cox, 1997). While RecA has a regulatory function acting as a coprotease in the cleavage of LexA in the SOS response, in recombinational events RecA itself plays the principal role by catalyzing two key reactions of homologous recombination: the pairing of homologous DNA molecules and the initiation of strand exchange. Since the interaction of the RecA protein with DNA is fundamental to its ability to catalyze homologous pairing Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. E-mail address of the corresponding author: [email protected] 0022-2836/99/091097±10 $30.00/0
and to act as a coprotease, the de®nition and characterization of the DNA binding domains of RecA are essential steps towards unraveling its molecular mechanisms (Takahashi et al., 1996). In the initial step of the pairing reaction, RecA binds to single-stranded DNA (ssDNA) and the nucleotide cofactor ATP, forming a nucleoprotein ®lament that is the homology-searching moiety mediating pairing with a double-stranded target DNA. On the basis of crystallographic studies, two disordered loops, L1 (amino acid positions 157164) and L2 (amino acid positions 195-209), facing the central cavity of the RecA ®lament (Figure 1) were proposed to be involved in DNA binding (Story et al., 1992). The structures of these loops could not be resolved in the X-ray study which was carried out in the absence of DNA and ATP, suggesting that these loops are mobile or of varying conformations. Experimental evidence showing that loop L2 is a DNA binding site has been increasing steadily: (i) proteolytic digestion of ssDNA-RecA complexes revealed a unique 4 kDa
1098
Saturation Mutagenesis of the RecA DNA Pairing Domain
Figure 1. (a) An a-carbon model of the RecA monomer with residues 157-164 (loop L1) and 193-212 (loop L2) in red. (b) The view along the axis of the RecA ®lament with loops L1 (amino and carboxyl termini are shown in white) and L2 (termini are in pink) projecting towards the central cavity, through which DNA is expected to pass.
peptide spanning L2 that is protected by the DNA (Gardner et al., 1995). (ii) The ¯uorescence of a tryptophan residue inserted at position 203, substituting for the central phenylalanine in loop L2, is quenched in RecA-DNA complexes (Maraboeuf et al., 1995). (iii) Photocross-linking studies demonstrated cross-links between DNA containing 5-iodouracil and amino acid residues Met164 and Phe203 loops L1 and L2, respectively (Malkov & Camerini-Otero, 1995; Wang & Adzuma, 1996). (iv) A loop L2 derived synthetic peptide not only binds to DNA, but can also promote the homologous pairing reaction (Voloshin et al., 1996). A comprehensive structural and functional characterization of this region, comprising at least part of the active site of RecA responsible for DNA binding and pairing, should elucidate the essential residues and structural constraints of the loop. In addition, such an analysis could also provide insight into the overall function and design of this catalytic domain and result in valuable information for the design of peptides with RecA-like activities. Here, we performed saturation mutagenesis of the amino acid residues 193-212 spanning the loop L2 region of Escherichia coli RecA, creating all possible 380 single mutants. We demonstrate that the disordered mobile loop L2 region is very sensitive to substitutions for most of its highly conserved residues. The results are discussed in terms of possible biochemical functions for individual residues as well as for the entire loop L2 region.
In order to assess the expression level of RecA protein in the cells carrying the plasmids encoding the mutant recA genes, we performed ELISA and Western blot analyses for all the mutants. Western blotting veri®ed that all the mutant RecA proteins
Results and Discussion
Figure 2. Using 20 synthetic oligonucleotides with degenerate codons targeting each amino acid position, respectively, resulted in an average recovery of 15 different point mutants of a given residue in the ®rst round of mutagenesis. The remaining mutants were created by using speci®c oligonucleotides in the second round. After all mutants had been obtained, their sequences were veri®ed for a second time before being assayed for their in vivo activity in the l plaque and the mitomycin C and UV resistance assays.
Here, we performed saturation mutagenesis in a region of the E. coli RecA protein that encompasses the disordered mobile loop L2 (residues 195-209) and its ¯anking residues (193, 194, 210, 211, and 212). The strategy used in the construction and characterization of the 380 recA mutants are outlined in Figure 2.
Saturation Mutagenesis of the RecA DNA Pairing Domain
were recognized speci®cally by the anti-RecA antibody, migrated according to their expected molecular size and showed similar levels of expression upon quantitation (data not shown). Quanti®cation by ELISA analysis revealed that the expression level of 83.9 % of the mutants was found to be within one (and 99.4 % were within two) standard deviation of the wild-type level of RecA expression from the pBluescript plasmid in a recAÿ strain. Most importantly, the expression level of the mutants did not correlate directly with their RecA activity. That is, as seen in Figure 3(a), some inactive mutants were expressed at high levels and vice versa. The high degree of conservation of the loop L2 region in prokaryotic RecAs has suggested very stringent requirements for most of its residues. In a sequence comparison between 64 eubacterial RecAs (Karlin & Brocchieri, 1996; Roca & Cox, 1997), 11 of the residues from 193 to 212 are identical, or nearly so, in all species, while six residues are highly conserved chemically (Table 1). Thus, 17 out of 20 residues are either identical or chemically conserved. The three exceptions are residues Met197, Asn205 and Thr210. However, any substitution at position 197 (often Glu or Gln) or 210 (often Pro) is always paired with at least one other substitution in loop L2 (Karlin & Brocchieri, 1996). Substitutions at Asn205 are often paired with other substitutions in L2 (interestingly, many times Glu at 197). We cannot rule out, however, that other changes elsewhere in RecA compensate for changes at residue 205. Positions 193, 194, 211 and 212, which ¯ank loop L2, as well as a central aromatic amino acid at position 203, are highly conserved among eukaryotic RecA homologs (Brendel et al., 1997). The recombination activity of each individual mutant was determined using three in vivo assays that assess the pro®ciency of RecA for homologous recombination and recombinational repair of damaged DNA. The ®rst assay measured the ability of RecA mutants expressed from a plasmid to allow a redgam l bacteriophage to form plaques (Enquist & Skalka, 1973). In l, concatamers of the phage genome have to form before processing, packaging, and lytic growth can occur. The redgam l phage cannot form concatamers from rolling circle replication because the gam protein is not present to protect its DNA from the RecBCD exonuclease. Furthermore, concatamers resulting from recombination of the circular l genomes cannot occur due to the absence of the red protein or a functional host RecA. Thus, the only way this mutant phage can lyse a RecA defective host is through the recombination activities of a RecA exogenously introduced. We also used two assays to examine the ability of our mutant RecAs to induce the SOS response and mediate the repair of double-stranded DNA (dsDNA) breaks. The ®rst used mitomycin C, and the second used UV irradiation to induce DNA damage. Results from all three assays were compiled for all 380 point
1099 mutants of the loop L2 region and compared to plasmid-encoded, wild-type RecA. The data obtained from the l plaque and UV and mitomycin C resistance assays are shown in Figure 3(a) and (b), respectively. While the l plaque assay tests for recombination pro®ciency only, the UV and mitomycin C resistance assays are measures of the recombinational repair of damaged DNA. Even though it has been shown that cell survival following DNA damage by UV or mitomycin C is more of a test for the recombinational pro®ciency of RecA than for its ability to induce the SOS response as a coprotease of LexA (Quillardet et al., 1982; Wang & Smith, 1986), the recombinational repair of damaged DNA is likely to be a more complex sequence of events than recombination alone. We therefore predicted and observed a higher stringency for functional substitutions in the UV and mitomycin C resistance assays (which led to almost identical results) than in the l plaque assay. Only three exceptions were found to this rule, V201C, V201N and T210P. That no substitutions are tolerated in our study at residues Asn193, Gln194, Arg196, Thr209, Gly211, Gly212, and only one substitution for Glu207, indicates that rather strict steric and chemical constraints are in effect at these positions, while the other residues that are almost strictly conserved Lys198, Gly200, Gly204 and Pro206 (Table 1) can be replaced by several other amino acid residues in the recombination assay. Interestingly, only Arg can substitute for Lys198 in the recombinational repair assay (Figure 3(b)). Among the six chemically conserved positions, only a few substitutions are functional in place of Ile195, Ile199, and Thr208, whereas positions Val201, Met202 and Phe203 are highly tolerant to mutations. Generally, the central part of loop L2 (amino acid residues 200-204) appears to be more tolerant to mutations than the ¯anking regions. Selected residues are discussed below. Asn193 did not tolerate any amino acid substitution. The inactivity of the mutants carrying a Gln or the negatively charged isosteric Asp at position Asn193 suggests that the precise positioning of this uncharged residue is important for RecA function. This may be due to the fact that not only is this residue part of strand 5 of the eight-stranded b-sheet in RecA (Story et al., 1992), but it also is most likely interacting via hydrogen bonds with the side-chain of Thr210 in the ¯ank of L2. This appears to be an extension of hydrophobic interactions between Phe215 and Leu191 in the ¯anks of L2 (Figure 4). Residue Gln194 has been proposed to interact with the g-phosphate of ATP, causing a change in conformation and stabilizing loop L2 or the preceding helix F in a conformation with high af®nity for DNA binding. ATP hydrolysis would resolve this interaction and return loop L2 to a conformation with low af®nity for DNA (Story & Steitz, 1992). Recently, a number of substitutions at this position were shown to be deleterious to recombi-
1100
Saturation Mutagenesis of the RecA DNA Pairing Domain
Figure 3. Summary of the in vivo phenotypes of all mutants. The results of the l plaque assay are shown in (a) The mutants were grouped into three categories: recA, recA/ÿ and recAÿ. The wild-type amino acid sequence of the loop L2 region is listed horizontally from 193 to 212, and the substitutions are listed from top to bottom in the order of decreasing b-sheet-forming propensity (Kim & Berg, 1993). The expression level (1-5) of the individual mutant proteins, as determined by ELISA, is indicated in the respective squares; level 1; outside 2 standard deviations (sd) below mean; level 2, between 2 sd and 1 sd below mean; level 3, within 1 sd below mean; level 4; within 1 sd above mean; level 5, between 1 sd and 2 sd above mean. The results of the UV and mitomycin resistance assays were combined and are presented in (b). The results were interpreted as recA, wild type activity; recA/ÿ, less active than wild type; recAÿ/, less active than recA/ÿ but more active than recAÿ; and recAÿ, inactive.
Saturation Mutagenesis of the RecA DNA Pairing Domain
1101
Table 1. Conservation of L2 region among 64 eubacterial RecAs Position 193N 194Q 195I 196R 197M 198K 199I 200G 201V 202M 203F 204G 205N 206P 207E 208T 209T 210T 211G 212G
Identical residues 63 64 45 64 40 63 54 64 61 59 54 63 38 64 64 61 64 53 64 63
Chemically conserved
Not conserved K(1)
L(15), V(4) L(1) R(1) V(8), M(1)
E(19), Q(3), D(1)
I(2) I(1), V(1) Y(8) T(1)
A(1) T(3) M(1) P(1) S(24), R(1)
S(1)
V(2) P(10), S(1)
A(1)
This table is a summary of data compiled by Karlin & Brocchieri (1996) and Roca & Cox (1997). A total of 11 out of the 20 residues are almost completely conserved, 64 or 63 sequences are identical and are in bold. Six other residues show only chemically conservative substitutions. Three residues Met197, Asn205 and Thr210 are not as highly conserved (but see text).
national repair and LexA cleavage (Kelley & Knight, 1997). This study showed that mutations Q194A, Q194E, and Q194N inhibited the formation of a high-af®nity ssDNA binding state when ATP was bound. It was proposed that Gln194 is an allosteric switch for the ATP-induced activation of RecA. Our results show that no amino acid could functionally replace this residue, and con®rm the necessity for this particular side-chain at precisely this location. Our experiments carried out with puri®ed mutant proteins demonstrated that Gln194, along with Argl96, is involved in the chemical steps of ATP hydrolysis (O.N.V., L. Wang & R. D. C.-O., unpublished results). Any substitution at position Arg196 resulted in the total inactivity of RecA as determined in the three in vivo assays. These results and, in particular, the non-functional Lys substitution suggests that not only a positive charge but also the guanidinium group of the Arg side-chain is necessary for RecA function. A possible role of Arg196 in the mutual regulation of DNA and ATP binding is discussed in more detail below. For Lys198, the one and only reported substitution found among other eubacteria is Arg (see Table 1). This amino acid (Arg) was found to be the only functional substitute for Lys in the assays scoring for recombinational repair, indicating the importance of the positively charged side-chain. In the l plaque assay, however, inhibition of RecA activity resulted from mutations only to negatively charged side-chains, to non-polar aromatic residues, as well as to Cys and Pro.
Figure 4. Putative interaction between Asn193 and Thr210. Coordinates were taken from the molecular structure of RecA (Story et al., 1992). Note that all the residues shown are not in loop L2 proper, but are in the ¯anks of L2.
With the exception of a minimally active Trp substitution, all substitutions for the completely conserved residue Glu207 abrogated RecA function. The inactive phenotypes of the Asp and the isosteric Gln mutations suggest that both the presence and the precise positioning of the negative charge are critical to RecA function. This is in concordance with the previously published inactivity of the E207Q (RecA659) protein for strand exchange (Cazaux et al., 1994). Gly211 and Gly212 are ¯anking the loop L2 and are the most highly conserved among eubacteria, as well as among eukaryotic RecA homologues (Brendel et al., 1997; Karlin & Brocchieri, 1996; Roca & Cox, 1997). Any substitution at these positions completely inactivates RecA. A very important role for the rotational ¯exibility of the peptide backbone at these positions is suggested by the observation that even Ala substitutions at either position lead to absolutely non-functional RecA proteins (Cazaux et al., 1994; Larminat et al., 1992). Story et al. (1992) hypothesized that residues 211 and 212 could be either directly involved in DNA binding or be playing a structural part in mediating the ATP-induced conformational change. DNA cross-linking (Malkov & Camerini-Otero, 1995) and ¯uorescence (Maraboeuf et al., 1995) studies have shown that residue Phe203 is part of or in very close proximity of the DNA binding site. In the l plaque assay most substitutions showed full RecA activity, while most substitutions had an inhibitory effect on recombinational repair activities. Gly204 is among those residues that are strictly conserved (Table 1), and this position shows sig-
1102 ni®cant sensitivity to mutation. Consistent with previously published results for G204A (RecA604), G204S (RecA430) and G204V (RecA605; Cazaux et al., 1991), partial activity was seen for substitutions with a group of amino acid residues having rather different chemical properties. Overall, steric constraints appear to be more important than rotational ¯exibility and side-chain information at this position. Pro206 is the only completely conserved residue that tolerates any substitution with no or little effect on RecA activity in the l plaque assay. The two obvious possibilities to explain this observation are that this residue is important in a much more subtle manner than can be detected by our assays, or that it plays a role in another activity of RecA. Similar considerations might apply to Gly200 and Gly204, residues that are also highly conserved yet tolerate a large degree of substitutions. Since neither a RecA-DNA nor a RecA-ATP cocrystal has yet been analyzed, not much is known about the active RecA-ATP-DNA conformation of loop L2. Experiments measuring the circular dichroism (CD) spectra of the 20 amino acid peptide corresponding to the loop L2 region showed that binding to DNA induced a conformational transition of the peptide from a random coil to a predominantly b-structure (Voloshin et al 1996; Wang et al., 1998). This conformational change seems to be important for binding to ssand dsDNA and unstacking of ssDNA, and was suggested to result from intrapeptide as well as interpeptide interactions (Voloshin et al., 1996; Wang et al., 1998). We have previously argued that the loop L2 region in the context of the whole RecA protein also assumes a b-structure upon binding to DNA and that this structure represents the active conformation of the loop L2 in the whole protein (Wang et al., 1998; O.N.V., L. Wang & R.D. C.-O., unpublished results). Four of the central residues in L2, Ile195, Met197, Ile199, and Phe203, tolerated a number of substitutions of differing chemical character and size as long as they possessed a relatively high propensity to form a b-structure (Chou & Fasman, 1978; Kim & Berg, 1993; Minor & Kim, 1994; Figure 3). Thus, the data from this study strongly suggest that the formation of a b-structure within loop L2 is crucial for the function of RecA. Combining the results of this study with our knowledge about the properties of the loop L2 peptide and previously known biochemical and genetic data about some point mutants (Cazaux et al., 1991, 1994; Voloshin et al., 1996; O.N.V., L. Wang & R.D. C.-O., unpublished results) we speculate that the loop L2 region is a minimal functional region for RecA that links DNA and ATP binding, as well as DNA pairing and ATPase activity. Although we know that this region can bind and pair DNAs (Voloshin et al., 1996), our proposal on the ATP binding and ATPase activities of L2 are mainly based on comparing this region
Saturation Mutagenesis of the RecA DNA Pairing Domain
with respect to the known structure and function relationships of GTPases. Intensive biochemical and structural investigation of G-proteins led to the deciphering of the molecular mechanisms of GTP hydrolysis and Gproteins activation by helper proteins. These studies revealed that two amino acid residues, Arg and Gln, are an invariant part of the GTPase active site (Sprang, 1997a,b). If the G-protein lacks arginine in the active site, this catalytic residue is provided in trans by a GTPase activating protein (Noel, 1997; Scheffzek et al., 1997). The indispensable character of Gln194 and Arg196, demonstrated here, strongly suggests that these two amino acid residues may play a key role in the hydrolysis of ATP by the RecA protein. Indeed, biochemical experiments performed with puri®ed mutant proteins con®rmed that the ability of the RecA protein to hydrolyze ATP and be activated by ATP and DNA completely relies on the presence of Gln194 and Arg196 (O.N.V., L. Wang & R.D. C.-O., unpublished results). It is worth noting that the active sites of GTP hydrolyzing proteins also contain invariant threonine and lysine residues, e.g. Thr181 and Lys46 in the case of Gia1 (Coleman et al., 1994). Thus, we propose that the invariant Thr209 and Lys198 (Arg198) of RecA might also be part of the ATP hydrolyzing pocket. Currently, the eukaryotic proteins Rad51 and Dmc1 are widely believed to be the functional analogs of RecA. These proteins share sequence homology, especially within a 230 amino acid residue core (Brendel et al., 1997). Furthermore, they form ®laments on DNA that are very similar to those formed by RecA (Benson et al., 1994; Ogawa et al., 1993), and there is an increasing ¯ow of data that shows that they carry out several of the hallmark biochemical reactions that are promoted by RecA (Benson et al., 1998; Li et al., 1997; New et al., 1998; Shinohara & Ogawa, 1998; Sung, 1994; Sung & Stratton, 1996). Although loop L2 is within the homologous core, and the amino acid residues that anchor the loop are invariant (Asn-Gln on the amino side, the central aromatic amino acid and Gly-Gly on the carboxyl end), some of the critical amino acid residues in L2 of RecA, such as Arg196 and Lys198, are missing in the corresponding region of Rad51 and Dmc1 (Story et al., 1993). Although these residues could in principle be provided to the catalytic pocket for ATP hydrolysis from adjacent regions in these proteins, we would like to speculate that they are provided in trans by other, as yet unidenti®ed, proteins. We base this speculation on the fact that both Rad51 and Dmc1 have kcat values for ATP hydrolysis that are from one to two orders of magnitude lower than the corresponding kcat value for RecA (Li et al., 1997; Sung, 1994; Sung & Stratton, 1996), and that the eukaryotic proteins are known to form protein-protein contacts with several other proteins (Benson et al., 1998; Buchhop et al., 1997; Clever et al., 1997; Golub et al., 1997; Hays et al.,
1103
Saturation Mutagenesis of the RecA DNA Pairing Domain
1995; Johnson & Symington, 1995; New et al., 1998; Scully et al., 1997; Shen et al., 1996; Shinohara & Ogawa, 1998; Sturzbecher et al., 1996; Sung, 1997). This is a situation reminiscent of that for certain GTPases, such as Ras, where the rate of hydrolysis of GTP is accelerated several orders of magnitude by heterodimer formation with GTPase-activating proteins (6APs) that provide the catalytic residues to stabilize the transition state of the GTPase reaction (Mittal et al., 1996). In other G-proteins, the intrinsic rate of GTP hydrolysis is already suf®ciently high (similar in some cases to that of RecA) and in those cases, as in RecA, all the catalytic residues are in cis. The identi®cation of those amino acid residues in loop L2 and its ¯ank (Asn193, Gln194, Arg196, Lys198, Glu207, Thr209, Gly211 and Gly212) that are essential for activity in vivo could delimit the sequence space that should be examined to engineer a peptide miniRecA with even better homologous pairing activities than the peptides already used by us (Voloshin et al., 1996), or perhaps even than the whole RecA protein, using approaches such as the recently published computational procedure by Dahiyat & Mayo (1997) or more traditional in vitro selection approaches, such as peptide phage display libraries (Cortese et al., 1996; McGregor, 1996; Schatz, 1994). Finally, knowing the molecular determinants of the loop L2 catalytic core (Arg196, Lys198, Glu207 and Thr209), residues presumably involved in binding to DNA and ATP, allows us to examine whether just a few amino acid residues may be used to sculpt the framework of the L2 b-loop. This analysis complements approaches used by other groups to investigate reduced amino acid alphabets to build functional peptides and proteins (Kamtekar et al., 1993; Regan & DeGrado, 1988; Riddle et al., 1997). Particularly noteworthy was the report by Baker and co-workers (Riddle et al., 1997) that showed that a small b-sheet protein, the 57 residue src SH3 domain, can be largely encoded using a ®ve letter amino acid alphabet: K, E, I, A and G. Assuming that our single point mutations would, for the most part, behave independently of each other, we have asked whether our mutagenesis results support such a simpli®ed alphabet. Since we know that the b-structure of loop L2 is suf®cient for homologous pairing (Voloshin et al., 1996; Wang et al., 1998) and we cannot exclude that speci®c contacts between L2 with either ligands or other parts of RecA are important for recombinational repair, we have focused only on the recombination activity of the mutants. If we exclude the highly conserved and charged amino acid residues (Arg196, Lys198, Glu207 and Thr209), we can clearly see that the three letter alphabet for non-charged amino acids (I, A and G) successfully used for the src SH3 domain is suf®cient. Interestingly, for our L2 b-loop, albeit a short b-loop, a different two letter alphabet for noncharged amino acids (T and M) appears to suf®ce.
Whether this even more simpli®ed alphabet might be of general use or even of use in designing miniRecA peptides remains to be proven. In conclusion, this study for the ®rst time demonstrates the importance of many individual residues in loop L2 for RecA function in vivo. Analyses of the 380 mutants resulted in a comprehensive set of genetic data that provides insight into the steric and chemical requirements at all 20 amino acid positions, delineates the conformational constraints of the loop L2 region, and offers a rationale to speculate about the role of single residues or the concerted action of several residues in the binding of DNA and ATP. Finally, these results represent the cornerstone for the design of RecA-like proteins or peptides. Since we have shown that 20 amino acid peptides derived loop L2 function in great part as a miniRecA in vitro (Voloshin et al., 1996), it is possible to actually test the activity of de novo designed peptides.
Materials and Methods Strains and plasmids XL1-Blue strain (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F0 proAB LaclqZM15 Tn10(Tetr)]) was used for plasmid construction and propagation, and for in vivo assays of the recombinational activities of all plasmid-borne recA mutants. The CJ236 strain and the helper phage M13K07 were used in generation of singlestranded uracil-substituted DNA template for the mutagenesis. The phages lZAPII (Stratagene) and lRX (constructed by deleting the lred and lgam genes from lZAPII, S. Pati and R.D. Camerini-Otero, unpublished results) were used for a l plaque assay that is dependent on RecA function. The recA wild-type gene was cloned into the polylinker region of pBluescript II SK() phagemid (Stratagene) and all mutant recA genes in this work are derivatives from that construct. Site-directed mutagenesis The Muta-Gene Phagemid In Vitro Mutagenesis Kit Version 2 (BioRad) was used, and mutagenesis was carried out according to the manufacturer's instructions. Mutations were introduced at each codon of the RecA loop L2 region (amino acid positions 193-212) such that each mutant carries a single amino acid substitution and that all possible amino acid substitutions were introduced at each position, resulting in 380 mutants. Amino acid substitutions were veri®ed by DNA sequencing of the loop L2 region plus approximately 50 ¯anking residues at each end. DNA sequencing was carried out using either a sequencing kit (Sequenase Version 2.0, United States Biochemicals) or with the ABI Model 373 automated sequencer. After the panel of 380 mutants was created, all clones were resequenced once more to eliminate/exclude all possible mistakes. Determination of cellular protein levels Protein lysates were prepared from each mutant, XL1Blue, XL1-Blue transformed with pBluescriptIISK (), and XL1-Blue carrying the pBluescriptIISK () plasmid with wild-type recA (WT), respectively. Overnight cul-
1104 tures were diluted 1:100 in LB-ampicillin and incubated at 37 C until the culture reached an A600 of 0.7. At this reading, 1.5 ml of the culture was pelleted, resuspended in 100 ml of distilled water and stored at ÿ70 C. Serum containing polyclonal antibodies against RecA (rabbit anti-RecA) was prepared by Hazelton Research Products. Western blots were performed as described (Sambrook et al., 1989) using PVDF membranes (NOVEX). The secondary antibody was linked to alkaline phosphatase, which was detected and quantitated using the Vistra ECF Western blotting system (Amersham) and the STORM imaging system (Molecular Dynamics). ELISAs were performed in Costar's 96-well plates using 2.5 mg of total protein per well and each plate was set up in triplicate. The color change was measured photometrically at 405 nm with a Titertek Multiscan PLUS MKII reader. After background subtraction, the values obtained in triplicate were averaged and used to determine the mean and standard deviation of each mutant relative to the control lysate from XL1-Blue carrying pBluescriptIISK (), expressing wildtype recA. Measurement of RecA activity in vivo Each mutant RecA in this study was tested in three in vivo assays for its ability to carry out both homologous recombination and recombinational repair of damaged DNA. Pro®ciency in homologous recombination was assessed using a l plaque assay. The ability of the redÿgamÿ l phage, lRX (S. Pati and R.D. C.-O., unpublished results), to support plaque formation on bacterial lawns is entirely dependent on host RecA function, and we determined the ef®ciency of plaque formation on lawns of recA mutants (Enquist & Skalka, 1973). Each E. coli XL1-Blue (recAÿ) subclone harboring the plasmidborne mutant RecA was grown from a single colony on selective media in 5 ml TB broth at 30 C overnight. The culture was pelleted, then resuspended in 10 mM MgSO4 to A600 0.5. A 200 ml sample of the cells were then infected with 500 pfu of phage lRX. After 15 minutes adsorption at 37 C, 2.5 ml of TB top agar was added to the infected cells, plated onto TB plates and incubated at 37 C overnight for the growth of the bacterial lawn and viral plaque formation. Positive and negative controls included XL1-Blue-carrying wild-type RecA in pBluescriptIISK() and XL1-Blue cells transformed with pBluescriptIISK(), respectively. The number of plaques for the recA mutants was scored in comparison with the positive control. The recombinational repair activity of the plasmidborne recA mutants was tested by observing cell survival following exposure to UV irradiation and mitomycin C. For both assays a cell culture of each mutant was grown to A600 0.5 and consequently diluted with culture media to A600 0.05. From these dilutions, 3ml each were spotted onto LB agar plates containing ampicillin (100 mg/ml) and on plates containing ampicillin (100 mg/ml) and mitomycin C for the UV resistance and varying concentrations of mitomycin C (0, 0.1, 0.2, 0.3 mg/ml) for the UV and mitomycin C resistance assays, respectively. Plates were then exposed to different dosages of short wave UV light (0, 0.2, 0.4, 0.8, 1.2, 1.8 and 3.0 mJ) using the ``energy'' mode of a Stratalinker 1800 equipped with a single 8W germicidal lamp. For comparison, the dose of 3 mJ is equivalent to irradiation by a lamp producing 600 mW/cm2 intensity of UV light from a distance of 17 cm for 11 seconds.
Saturation Mutagenesis of the RecA DNA Pairing Domain After incubation at 37 C overnight, the culture spots were evaluated in comparison with the positive and negative controls. The results were interpreted as recA wild type activity, recA/ÿ less active than wild type, recAÿ/ less active than recA/ÿ but more active than recAÿ, and recAÿ inactive.
Acknowledgments We thank Peggy Hsieh, Susan Gottesman, Kyoishi Mizuuchi and Rick Proia for reading the manuscript and helpful comments. We thank George Poy, Amit Rahman and Linda Robinson for their technical help.
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Saturation Mutagenesis of the RecA DNA Pairing Domain Gardner, R. V., Voloshin, O. N. & Camerini-Otero, R. D. (1995). The identi®cation of the single-stranded DNA-binding domain of the Escherichia coli RecA protein. Eur. J. Biochem. 233, 419-425. Golub, E. L., Kovalenko, O. V., Gupta, R. C., Ward, D. C. & Radding, C. M. (1997). Interaction of human recombination proteins Rad51 and Rad54. Nucl. Acids Res. 25, 4106-4110. Hays, S. L., Firmenich, A. A. & Berg, P. (1995). Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc. Natl Acad. Sci. USA, 92, 6925-6929. Johnson, R. D. & Symington, L. S. (1995). Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol. Cell. Biol. 15, 4843-4850. Karntekar, S., Schiffer, J. M., Xiong, H., Babik, J. M. & Hecht, M. H. (1993). Protein design by binary patterning of polar and nonpolar amino acids. Science, 262, 1680-1685. Karlin, S. & Brocchieri, L. (1996). Evolutionary conservation of RecA genes in relation to protein structure and function. J. Bacteriol. 178, 1881-1894. Kelley, J. A. & Knight, K. L. (1997). Allosteric regulation of RecA protein function is mediated by GIn194. J. Biol. Chem. 272, 25778-25782. Kim, C. A. & Berg, J. M. (1993). Thermodynamic b-sheet propensities measured using a zinc-®nger host peptide. Nature, 362, 267-270. Larminat, F., Cazaux, C., Germanier, M. & Defais, M. (1992). New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity. J. Bacteriol. 174, 6264-6269. Li, Z., Golub, E. I., Gupta, R. & Radding, C. M. (1997). Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein. Proc. Natl Acad. Sci. USA, 94, 11221-11226. Malkov, V. A. & Camerini-Otero, R. D. (1995). Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops LI (amino acid residues 157-164) and L2 (amino acid residues 195-209). J. Biol. Chem. 270, 30230-30233. Maraboeuf, F., Voloshin, O., Camerini-Otero, R. D. & Takahashi, M. (1995). The central aromatic residue in loop L2 of RecA interacts with DNA. Quenching of the ¯uorescence of a tryptophan reporter inserted in L2 upon binding to DNA. J. Biol. Chem. 270, 30927-30932. McGregor, D. (1996). Selection of proteins and peptides from libraries displayed on ®lamentous bacteriophage. Mol. Biotechnol. 6, 155-162. Minor, D. L., Jr & Kim, P. S. (1994). Measurement of the b-sheet-forming propensities of amino acids. Nature, 367, 660-663. Mittal, R., Ahmadian, M. R., Goody, R. S. & Wittinghofer, A. (1996). Formation of a transitionstate analog of the Ras GTPase reaction by RasGDP, tetra¯uoroaluminate, and GTPase-activating proteins. Science, 273, 115-117. New, J. H., Sugiyama, T., Zaitseva, E. & Kowalczykowski, S. C. (1998). Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature, 391, 407-410. Noel, J. P. (1997). Turning off the Ras switch with the ¯ick of a ®nger. Nature Struct. Biol. 4, 677-680. Ogawa, T., Yu, X., Shinohara, A. & Egelman, E. H. (1993). Similarity of the yeast Rad51 ®lament to the bacterial RecA ®lament. Science, 259, 1896-1899.
1105 Quillardet, P., Huisman, O., D'Ari, R. & Hofnung, M. (1982). The SOS chromotest: direct assay of the expression of gene s®A as a measure of genotoxicity of chemicals. Biochimie, 64, 797-801. Regan, L. & DeGrado, W. F. (1988). Characterization of a helical protein designed from ®rst principles. Science, 241, 976-978. Riddle, D. S., Santiago, J. V., Bray-Hall, S. T., Doshi, N., Grantcharova, V. P., Yi, Q. & Baker, D. (1997). Functional rapidly folding proteins from simpli®ed amino acid sequences. Nature Struct. Biol. 4, 805809. Roca, A. I. & Cox, M. M. (1997). RecA protein: structure, function, and role in recombinational DNA repair. Prog. Nucl. Acid Res. Mol. Biol. 56, 129-223. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edit., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schatz, P. J. (1994). Construction and screening of biological peptide libraries. Curr. Opin. Biotechnol. 5, 487-494. Schef®ek, K., Ahmadian, M. R., Kabsch, W., WiesmuÈller, L., Lautwein, A., Schmitz, F. & Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science, 277, 333-338. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. & Livingston, D. M. (1997). Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell, 88, 265-275. Shen, Z., Cloud, K. G., Chen, D. J. & Park, M. S. (1996). Speci®c interactions between the human RAD51 and RAD52 proteins. J. Biol. Chem. 271, 148-152. Shinohara, A. & Ogawa, T. (1998). Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature, 391, 404-407. Sprang, S. R. (1997a). G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639-678. Sprang, S. R. (1997b). G proteins, effectors and GAPs: structure and mechanism. Curr. Opin. Struct. Biol. 7, 849-856. Story, R. M. & Steitz, T. A. (1992). Structure of the recA protein-ADP complex. Nature, 355, 374-376. Story, R. M., Weber, I. T. & Steitz, T. A. (1992). The structure of the E. coli recA protein monomer and polymer. Nature, 355, 318-325. Story, R. M., Bishop, D. K., Kleckner, N. & Steitz, T. A. (1993). Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast. Science, 259, 1892-1896. Sturzbecher, H. W., Donzelmann, B., Henning, W., Knippschild, U. & Buchhop, S. (1996). p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J. 15, 19922002. Sung, P. (1994). Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science, 265, 1241-1243. Sung, P. (1997). Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 11, 1111-1121. Sung, P. & Stratton, S. A. (1996). Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. J. Biol. Chem. 271, 2798327986.
1106
Saturation Mutagenesis of the RecA DNA Pairing Domain
Takahashi, M., Maraboetif, F. & Norden, B. (1996). Locations of functional domains in the RecA protein. Overlap of domains and regulation of activities. Eur. J. Biochem. 242, 20-28. Voloshin, O. N., Wang, L. & Camerini-Otero, R. D. (1996). Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA. Science, 272, 868-872. Wang, L., Voloshin, O. N., Stasiak, A., Stasiak, A. & Camerini-Otero, R. D. (1998). Homologous DNA pairing domain peptides of RecA protein: intrinsic
propensity to form b-structure and ®laments. J. Mol. Biol. 277, 1-11. Wang, T. C. & Smith, K. C. (1986). recA (Srf) suppression of recF de®ciency in the postreplication repair of UV-irradiated Escherichia coli K-12. J. Bacteriol. 168, 940-946. Wang, Y. & Adzuma, K. (1996). Differential proximity probing of two DNA binding sites in the Escherichia coil recA protein using photo-cross-linking methods. Biochemistry, 35, 3563-3571.
Edited by M. Gottesman (Received 17 August 1998; received in revised form 27 November 1998; accepted 14 December 1998)
J. Mol. Biol. (1999) 286, 1097±1106
Saturation Mutagenesis of the E. coli RecA Loop L2 Homologous DNA Pairing Region Reveals Residues Essential for Recombination and Recombinational Repair Konstanze HoÈrtnagel, Oleg N. Voloshin, Hai H. Kinal, Ning Ma Carianne Schaffer-Judge and R. Daniel Camerini-Otero* Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda MD 20892-1810, USA
*Corresponding author
The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational pro®ciency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identi®ed as stringently required for recombinational events in bacterial cells. In addition, our ®ndings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming ®lamentous b-structures, we propose how the information from the mutant analysis might facilitate the use of a simpli®ed amino acid alphabet to design b-structure forming L2 peptides with improved RecAlike activities. Keywords: RecA protein; homologous recombination; recombinational repair; DNA pairing; ATPase activity
Introduction The prokaryotic RecA protein is an essential component of many cellular processes, including general homologous recombination, recombinational DNA repair, and induction of the SOS response to DNA damage (Dunderdale & West, 1994; Roca & Cox, 1997). While RecA has a regulatory function acting as a coprotease in the cleavage of LexA in the SOS response, in recombinational events RecA itself plays the principal role by catalyzing two key reactions of homologous recombination: the pairing of homologous DNA molecules and the initiation of strand exchange. Since the interaction of the RecA protein with DNA is fundamental to its ability to catalyze homologous pairing Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. E-mail address of the corresponding author: [email protected] 0022-2836/99/091097±10 $30.00/0
and to act as a coprotease, the de®nition and characterization of the DNA binding domains of RecA are essential steps towards unraveling its molecular mechanisms (Takahashi et al., 1996). In the initial step of the pairing reaction, RecA binds to single-stranded DNA (ssDNA) and the nucleotide cofactor ATP, forming a nucleoprotein ®lament that is the homology-searching moiety mediating pairing with a double-stranded target DNA. On the basis of crystallographic studies, two disordered loops, L1 (amino acid positions 157164) and L2 (amino acid positions 195-209), facing the central cavity of the RecA ®lament (Figure 1) were proposed to be involved in DNA binding (Story et al., 1992). The structures of these loops could not be resolved in the X-ray study which was carried out in the absence of DNA and ATP, suggesting that these loops are mobile or of varying conformations. Experimental evidence showing that loop L2 is a DNA binding site has been increasing steadily: (i) proteolytic digestion of ssDNA-RecA complexes revealed a unique 4 kDa
1098
Saturation Mutagenesis of the RecA DNA Pairing Domain
Figure 1. (a) An a-carbon model of the RecA monomer with residues 157-164 (loop L1) and 193-212 (loop L2) in red. (b) The view along the axis of the RecA ®lament with loops L1 (amino and carboxyl termini are shown in white) and L2 (termini are in pink) projecting towards the central cavity, through which DNA is expected to pass.
peptide spanning L2 that is protected by the DNA (Gardner et al., 1995). (ii) The ¯uorescence of a tryptophan residue inserted at position 203, substituting for the central phenylalanine in loop L2, is quenched in RecA-DNA complexes (Maraboeuf et al., 1995). (iii) Photocross-linking studies demonstrated cross-links between DNA containing 5-iodouracil and amino acid residues Met164 and Phe203 loops L1 and L2, respectively (Malkov & Camerini-Otero, 1995; Wang & Adzuma, 1996). (iv) A loop L2 derived synthetic peptide not only binds to DNA, but can also promote the homologous pairing reaction (Voloshin et al., 1996). A comprehensive structural and functional characterization of this region, comprising at least part of the active site of RecA responsible for DNA binding and pairing, should elucidate the essential residues and structural constraints of the loop. In addition, such an analysis could also provide insight into the overall function and design of this catalytic domain and result in valuable information for the design of peptides with RecA-like activities. Here, we performed saturation mutagenesis of the amino acid residues 193-212 spanning the loop L2 region of Escherichia coli RecA, creating all possible 380 single mutants. We demonstrate that the disordered mobile loop L2 region is very sensitive to substitutions for most of its highly conserved residues. The results are discussed in terms of possible biochemical functions for individual residues as well as for the entire loop L2 region.
In order to assess the expression level of RecA protein in the cells carrying the plasmids encoding the mutant recA genes, we performed ELISA and Western blot analyses for all the mutants. Western blotting veri®ed that all the mutant RecA proteins
Results and Discussion
Figure 2. Using 20 synthetic oligonucleotides with degenerate codons targeting each amino acid position, respectively, resulted in an average recovery of 15 different point mutants of a given residue in the ®rst round of mutagenesis. The remaining mutants were created by using speci®c oligonucleotides in the second round. After all mutants had been obtained, their sequences were veri®ed for a second time before being assayed for their in vivo activity in the l plaque and the mitomycin C and UV resistance assays.
Here, we performed saturation mutagenesis in a region of the E. coli RecA protein that encompasses the disordered mobile loop L2 (residues 195-209) and its ¯anking residues (193, 194, 210, 211, and 212). The strategy used in the construction and characterization of the 380 recA mutants are outlined in Figure 2.
Saturation Mutagenesis of the RecA DNA Pairing Domain
were recognized speci®cally by the anti-RecA antibody, migrated according to their expected molecular size and showed similar levels of expression upon quantitation (data not shown). Quanti®cation by ELISA analysis revealed that the expression level of 83.9 % of the mutants was found to be within one (and 99.4 % were within two) standard deviation of the wild-type level of RecA expression from the pBluescript plasmid in a recAÿ strain. Most importantly, the expression level of the mutants did not correlate directly with their RecA activity. That is, as seen in Figure 3(a), some inactive mutants were expressed at high levels and vice versa. The high degree of conservation of the loop L2 region in prokaryotic RecAs has suggested very stringent requirements for most of its residues. In a sequence comparison between 64 eubacterial RecAs (Karlin & Brocchieri, 1996; Roca & Cox, 1997), 11 of the residues from 193 to 212 are identical, or nearly so, in all species, while six residues are highly conserved chemically (Table 1). Thus, 17 out of 20 residues are either identical or chemically conserved. The three exceptions are residues Met197, Asn205 and Thr210. However, any substitution at position 197 (often Glu or Gln) or 210 (often Pro) is always paired with at least one other substitution in loop L2 (Karlin & Brocchieri, 1996). Substitutions at Asn205 are often paired with other substitutions in L2 (interestingly, many times Glu at 197). We cannot rule out, however, that other changes elsewhere in RecA compensate for changes at residue 205. Positions 193, 194, 211 and 212, which ¯ank loop L2, as well as a central aromatic amino acid at position 203, are highly conserved among eukaryotic RecA homologs (Brendel et al., 1997). The recombination activity of each individual mutant was determined using three in vivo assays that assess the pro®ciency of RecA for homologous recombination and recombinational repair of damaged DNA. The ®rst assay measured the ability of RecA mutants expressed from a plasmid to allow a redgam l bacteriophage to form plaques (Enquist & Skalka, 1973). In l, concatamers of the phage genome have to form before processing, packaging, and lytic growth can occur. The redgam l phage cannot form concatamers from rolling circle replication because the gam protein is not present to protect its DNA from the RecBCD exonuclease. Furthermore, concatamers resulting from recombination of the circular l genomes cannot occur due to the absence of the red protein or a functional host RecA. Thus, the only way this mutant phage can lyse a RecA defective host is through the recombination activities of a RecA exogenously introduced. We also used two assays to examine the ability of our mutant RecAs to induce the SOS response and mediate the repair of double-stranded DNA (dsDNA) breaks. The ®rst used mitomycin C, and the second used UV irradiation to induce DNA damage. Results from all three assays were compiled for all 380 point
1099 mutants of the loop L2 region and compared to plasmid-encoded, wild-type RecA. The data obtained from the l plaque and UV and mitomycin C resistance assays are shown in Figure 3(a) and (b), respectively. While the l plaque assay tests for recombination pro®ciency only, the UV and mitomycin C resistance assays are measures of the recombinational repair of damaged DNA. Even though it has been shown that cell survival following DNA damage by UV or mitomycin C is more of a test for the recombinational pro®ciency of RecA than for its ability to induce the SOS response as a coprotease of LexA (Quillardet et al., 1982; Wang & Smith, 1986), the recombinational repair of damaged DNA is likely to be a more complex sequence of events than recombination alone. We therefore predicted and observed a higher stringency for functional substitutions in the UV and mitomycin C resistance assays (which led to almost identical results) than in the l plaque assay. Only three exceptions were found to this rule, V201C, V201N and T210P. That no substitutions are tolerated in our study at residues Asn193, Gln194, Arg196, Thr209, Gly211, Gly212, and only one substitution for Glu207, indicates that rather strict steric and chemical constraints are in effect at these positions, while the other residues that are almost strictly conserved Lys198, Gly200, Gly204 and Pro206 (Table 1) can be replaced by several other amino acid residues in the recombination assay. Interestingly, only Arg can substitute for Lys198 in the recombinational repair assay (Figure 3(b)). Among the six chemically conserved positions, only a few substitutions are functional in place of Ile195, Ile199, and Thr208, whereas positions Val201, Met202 and Phe203 are highly tolerant to mutations. Generally, the central part of loop L2 (amino acid residues 200-204) appears to be more tolerant to mutations than the ¯anking regions. Selected residues are discussed below. Asn193 did not tolerate any amino acid substitution. The inactivity of the mutants carrying a Gln or the negatively charged isosteric Asp at position Asn193 suggests that the precise positioning of this uncharged residue is important for RecA function. This may be due to the fact that not only is this residue part of strand 5 of the eight-stranded b-sheet in RecA (Story et al., 1992), but it also is most likely interacting via hydrogen bonds with the side-chain of Thr210 in the ¯ank of L2. This appears to be an extension of hydrophobic interactions between Phe215 and Leu191 in the ¯anks of L2 (Figure 4). Residue Gln194 has been proposed to interact with the g-phosphate of ATP, causing a change in conformation and stabilizing loop L2 or the preceding helix F in a conformation with high af®nity for DNA binding. ATP hydrolysis would resolve this interaction and return loop L2 to a conformation with low af®nity for DNA (Story & Steitz, 1992). Recently, a number of substitutions at this position were shown to be deleterious to recombi-
1100
Saturation Mutagenesis of the RecA DNA Pairing Domain
Figure 3. Summary of the in vivo phenotypes of all mutants. The results of the l plaque assay are shown in (a) The mutants were grouped into three categories: recA, recA/ÿ and recAÿ. The wild-type amino acid sequence of the loop L2 region is listed horizontally from 193 to 212, and the substitutions are listed from top to bottom in the order of decreasing b-sheet-forming propensity (Kim & Berg, 1993). The expression level (1-5) of the individual mutant proteins, as determined by ELISA, is indicated in the respective squares; level 1; outside 2 standard deviations (sd) below mean; level 2, between 2 sd and 1 sd below mean; level 3, within 1 sd below mean; level 4; within 1 sd above mean; level 5, between 1 sd and 2 sd above mean. The results of the UV and mitomycin resistance assays were combined and are presented in (b). The results were interpreted as recA, wild type activity; recA/ÿ, less active than wild type; recAÿ/, less active than recA/ÿ but more active than recAÿ; and recAÿ, inactive.
Saturation Mutagenesis of the RecA DNA Pairing Domain
1101
Table 1. Conservation of L2 region among 64 eubacterial RecAs Position 193N 194Q 195I 196R 197M 198K 199I 200G 201V 202M 203F 204G 205N 206P 207E 208T 209T 210T 211G 212G
Identical residues 63 64 45 64 40 63 54 64 61 59 54 63 38 64 64 61 64 53 64 63
Chemically conserved
Not conserved K(1)
L(15), V(4) L(1) R(1) V(8), M(1)
E(19), Q(3), D(1)
I(2) I(1), V(1) Y(8) T(1)
A(1) T(3) M(1) P(1) S(24), R(1)
S(1)
V(2) P(10), S(1)
A(1)
This table is a summary of data compiled by Karlin & Brocchieri (1996) and Roca & Cox (1997). A total of 11 out of the 20 residues are almost completely conserved, 64 or 63 sequences are identical and are in bold. Six other residues show only chemically conservative substitutions. Three residues Met197, Asn205 and Thr210 are not as highly conserved (but see text).
national repair and LexA cleavage (Kelley & Knight, 1997). This study showed that mutations Q194A, Q194E, and Q194N inhibited the formation of a high-af®nity ssDNA binding state when ATP was bound. It was proposed that Gln194 is an allosteric switch for the ATP-induced activation of RecA. Our results show that no amino acid could functionally replace this residue, and con®rm the necessity for this particular side-chain at precisely this location. Our experiments carried out with puri®ed mutant proteins demonstrated that Gln194, along with Argl96, is involved in the chemical steps of ATP hydrolysis (O.N.V., L. Wang & R. D. C.-O., unpublished results). Any substitution at position Arg196 resulted in the total inactivity of RecA as determined in the three in vivo assays. These results and, in particular, the non-functional Lys substitution suggests that not only a positive charge but also the guanidinium group of the Arg side-chain is necessary for RecA function. A possible role of Arg196 in the mutual regulation of DNA and ATP binding is discussed in more detail below. For Lys198, the one and only reported substitution found among other eubacteria is Arg (see Table 1). This amino acid (Arg) was found to be the only functional substitute for Lys in the assays scoring for recombinational repair, indicating the importance of the positively charged side-chain. In the l plaque assay, however, inhibition of RecA activity resulted from mutations only to negatively charged side-chains, to non-polar aromatic residues, as well as to Cys and Pro.
Figure 4. Putative interaction between Asn193 and Thr210. Coordinates were taken from the molecular structure of RecA (Story et al., 1992). Note that all the residues shown are not in loop L2 proper, but are in the ¯anks of L2.
With the exception of a minimally active Trp substitution, all substitutions for the completely conserved residue Glu207 abrogated RecA function. The inactive phenotypes of the Asp and the isosteric Gln mutations suggest that both the presence and the precise positioning of the negative charge are critical to RecA function. This is in concordance with the previously published inactivity of the E207Q (RecA659) protein for strand exchange (Cazaux et al., 1994). Gly211 and Gly212 are ¯anking the loop L2 and are the most highly conserved among eubacteria, as well as among eukaryotic RecA homologues (Brendel et al., 1997; Karlin & Brocchieri, 1996; Roca & Cox, 1997). Any substitution at these positions completely inactivates RecA. A very important role for the rotational ¯exibility of the peptide backbone at these positions is suggested by the observation that even Ala substitutions at either position lead to absolutely non-functional RecA proteins (Cazaux et al., 1994; Larminat et al., 1992). Story et al. (1992) hypothesized that residues 211 and 212 could be either directly involved in DNA binding or be playing a structural part in mediating the ATP-induced conformational change. DNA cross-linking (Malkov & Camerini-Otero, 1995) and ¯uorescence (Maraboeuf et al., 1995) studies have shown that residue Phe203 is part of or in very close proximity of the DNA binding site. In the l plaque assay most substitutions showed full RecA activity, while most substitutions had an inhibitory effect on recombinational repair activities. Gly204 is among those residues that are strictly conserved (Table 1), and this position shows sig-
1102 ni®cant sensitivity to mutation. Consistent with previously published results for G204A (RecA604), G204S (RecA430) and G204V (RecA605; Cazaux et al., 1991), partial activity was seen for substitutions with a group of amino acid residues having rather different chemical properties. Overall, steric constraints appear to be more important than rotational ¯exibility and side-chain information at this position. Pro206 is the only completely conserved residue that tolerates any substitution with no or little effect on RecA activity in the l plaque assay. The two obvious possibilities to explain this observation are that this residue is important in a much more subtle manner than can be detected by our assays, or that it plays a role in another activity of RecA. Similar considerations might apply to Gly200 and Gly204, residues that are also highly conserved yet tolerate a large degree of substitutions. Since neither a RecA-DNA nor a RecA-ATP cocrystal has yet been analyzed, not much is known about the active RecA-ATP-DNA conformation of loop L2. Experiments measuring the circular dichroism (CD) spectra of the 20 amino acid peptide corresponding to the loop L2 region showed that binding to DNA induced a conformational transition of the peptide from a random coil to a predominantly b-structure (Voloshin et al 1996; Wang et al., 1998). This conformational change seems to be important for binding to ssand dsDNA and unstacking of ssDNA, and was suggested to result from intrapeptide as well as interpeptide interactions (Voloshin et al., 1996; Wang et al., 1998). We have previously argued that the loop L2 region in the context of the whole RecA protein also assumes a b-structure upon binding to DNA and that this structure represents the active conformation of the loop L2 in the whole protein (Wang et al., 1998; O.N.V., L. Wang & R.D. C.-O., unpublished results). Four of the central residues in L2, Ile195, Met197, Ile199, and Phe203, tolerated a number of substitutions of differing chemical character and size as long as they possessed a relatively high propensity to form a b-structure (Chou & Fasman, 1978; Kim & Berg, 1993; Minor & Kim, 1994; Figure 3). Thus, the data from this study strongly suggest that the formation of a b-structure within loop L2 is crucial for the function of RecA. Combining the results of this study with our knowledge about the properties of the loop L2 peptide and previously known biochemical and genetic data about some point mutants (Cazaux et al., 1991, 1994; Voloshin et al., 1996; O.N.V., L. Wang & R.D. C.-O., unpublished results) we speculate that the loop L2 region is a minimal functional region for RecA that links DNA and ATP binding, as well as DNA pairing and ATPase activity. Although we know that this region can bind and pair DNAs (Voloshin et al., 1996), our proposal on the ATP binding and ATPase activities of L2 are mainly based on comparing this region
Saturation Mutagenesis of the RecA DNA Pairing Domain
with respect to the known structure and function relationships of GTPases. Intensive biochemical and structural investigation of G-proteins led to the deciphering of the molecular mechanisms of GTP hydrolysis and Gproteins activation by helper proteins. These studies revealed that two amino acid residues, Arg and Gln, are an invariant part of the GTPase active site (Sprang, 1997a,b). If the G-protein lacks arginine in the active site, this catalytic residue is provided in trans by a GTPase activating protein (Noel, 1997; Scheffzek et al., 1997). The indispensable character of Gln194 and Arg196, demonstrated here, strongly suggests that these two amino acid residues may play a key role in the hydrolysis of ATP by the RecA protein. Indeed, biochemical experiments performed with puri®ed mutant proteins con®rmed that the ability of the RecA protein to hydrolyze ATP and be activated by ATP and DNA completely relies on the presence of Gln194 and Arg196 (O.N.V., L. Wang & R.D. C.-O., unpublished results). It is worth noting that the active sites of GTP hydrolyzing proteins also contain invariant threonine and lysine residues, e.g. Thr181 and Lys46 in the case of Gia1 (Coleman et al., 1994). Thus, we propose that the invariant Thr209 and Lys198 (Arg198) of RecA might also be part of the ATP hydrolyzing pocket. Currently, the eukaryotic proteins Rad51 and Dmc1 are widely believed to be the functional analogs of RecA. These proteins share sequence homology, especially within a 230 amino acid residue core (Brendel et al., 1997). Furthermore, they form ®laments on DNA that are very similar to those formed by RecA (Benson et al., 1994; Ogawa et al., 1993), and there is an increasing ¯ow of data that shows that they carry out several of the hallmark biochemical reactions that are promoted by RecA (Benson et al., 1998; Li et al., 1997; New et al., 1998; Shinohara & Ogawa, 1998; Sung, 1994; Sung & Stratton, 1996). Although loop L2 is within the homologous core, and the amino acid residues that anchor the loop are invariant (Asn-Gln on the amino side, the central aromatic amino acid and Gly-Gly on the carboxyl end), some of the critical amino acid residues in L2 of RecA, such as Arg196 and Lys198, are missing in the corresponding region of Rad51 and Dmc1 (Story et al., 1993). Although these residues could in principle be provided to the catalytic pocket for ATP hydrolysis from adjacent regions in these proteins, we would like to speculate that they are provided in trans by other, as yet unidenti®ed, proteins. We base this speculation on the fact that both Rad51 and Dmc1 have kcat values for ATP hydrolysis that are from one to two orders of magnitude lower than the corresponding kcat value for RecA (Li et al., 1997; Sung, 1994; Sung & Stratton, 1996), and that the eukaryotic proteins are known to form protein-protein contacts with several other proteins (Benson et al., 1998; Buchhop et al., 1997; Clever et al., 1997; Golub et al., 1997; Hays et al.,
1103
Saturation Mutagenesis of the RecA DNA Pairing Domain
1995; Johnson & Symington, 1995; New et al., 1998; Scully et al., 1997; Shen et al., 1996; Shinohara & Ogawa, 1998; Sturzbecher et al., 1996; Sung, 1997). This is a situation reminiscent of that for certain GTPases, such as Ras, where the rate of hydrolysis of GTP is accelerated several orders of magnitude by heterodimer formation with GTPase-activating proteins (6APs) that provide the catalytic residues to stabilize the transition state of the GTPase reaction (Mittal et al., 1996). In other G-proteins, the intrinsic rate of GTP hydrolysis is already suf®ciently high (similar in some cases to that of RecA) and in those cases, as in RecA, all the catalytic residues are in cis. The identi®cation of those amino acid residues in loop L2 and its ¯ank (Asn193, Gln194, Arg196, Lys198, Glu207, Thr209, Gly211 and Gly212) that are essential for activity in vivo could delimit the sequence space that should be examined to engineer a peptide miniRecA with even better homologous pairing activities than the peptides already used by us (Voloshin et al., 1996), or perhaps even than the whole RecA protein, using approaches such as the recently published computational procedure by Dahiyat & Mayo (1997) or more traditional in vitro selection approaches, such as peptide phage display libraries (Cortese et al., 1996; McGregor, 1996; Schatz, 1994). Finally, knowing the molecular determinants of the loop L2 catalytic core (Arg196, Lys198, Glu207 and Thr209), residues presumably involved in binding to DNA and ATP, allows us to examine whether just a few amino acid residues may be used to sculpt the framework of the L2 b-loop. This analysis complements approaches used by other groups to investigate reduced amino acid alphabets to build functional peptides and proteins (Kamtekar et al., 1993; Regan & DeGrado, 1988; Riddle et al., 1997). Particularly noteworthy was the report by Baker and co-workers (Riddle et al., 1997) that showed that a small b-sheet protein, the 57 residue src SH3 domain, can be largely encoded using a ®ve letter amino acid alphabet: K, E, I, A and G. Assuming that our single point mutations would, for the most part, behave independently of each other, we have asked whether our mutagenesis results support such a simpli®ed alphabet. Since we know that the b-structure of loop L2 is suf®cient for homologous pairing (Voloshin et al., 1996; Wang et al., 1998) and we cannot exclude that speci®c contacts between L2 with either ligands or other parts of RecA are important for recombinational repair, we have focused only on the recombination activity of the mutants. If we exclude the highly conserved and charged amino acid residues (Arg196, Lys198, Glu207 and Thr209), we can clearly see that the three letter alphabet for non-charged amino acids (I, A and G) successfully used for the src SH3 domain is suf®cient. Interestingly, for our L2 b-loop, albeit a short b-loop, a different two letter alphabet for noncharged amino acids (T and M) appears to suf®ce.
Whether this even more simpli®ed alphabet might be of general use or even of use in designing miniRecA peptides remains to be proven. In conclusion, this study for the ®rst time demonstrates the importance of many individual residues in loop L2 for RecA function in vivo. Analyses of the 380 mutants resulted in a comprehensive set of genetic data that provides insight into the steric and chemical requirements at all 20 amino acid positions, delineates the conformational constraints of the loop L2 region, and offers a rationale to speculate about the role of single residues or the concerted action of several residues in the binding of DNA and ATP. Finally, these results represent the cornerstone for the design of RecA-like proteins or peptides. Since we have shown that 20 amino acid peptides derived loop L2 function in great part as a miniRecA in vitro (Voloshin et al., 1996), it is possible to actually test the activity of de novo designed peptides.
Materials and Methods Strains and plasmids XL1-Blue strain (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F0 proAB LaclqZM15 Tn10(Tetr)]) was used for plasmid construction and propagation, and for in vivo assays of the recombinational activities of all plasmid-borne recA mutants. The CJ236 strain and the helper phage M13K07 were used in generation of singlestranded uracil-substituted DNA template for the mutagenesis. The phages lZAPII (Stratagene) and lRX (constructed by deleting the lred and lgam genes from lZAPII, S. Pati and R.D. Camerini-Otero, unpublished results) were used for a l plaque assay that is dependent on RecA function. The recA wild-type gene was cloned into the polylinker region of pBluescript II SK() phagemid (Stratagene) and all mutant recA genes in this work are derivatives from that construct. Site-directed mutagenesis The Muta-Gene Phagemid In Vitro Mutagenesis Kit Version 2 (BioRad) was used, and mutagenesis was carried out according to the manufacturer's instructions. Mutations were introduced at each codon of the RecA loop L2 region (amino acid positions 193-212) such that each mutant carries a single amino acid substitution and that all possible amino acid substitutions were introduced at each position, resulting in 380 mutants. Amino acid substitutions were veri®ed by DNA sequencing of the loop L2 region plus approximately 50 ¯anking residues at each end. DNA sequencing was carried out using either a sequencing kit (Sequenase Version 2.0, United States Biochemicals) or with the ABI Model 373 automated sequencer. After the panel of 380 mutants was created, all clones were resequenced once more to eliminate/exclude all possible mistakes. Determination of cellular protein levels Protein lysates were prepared from each mutant, XL1Blue, XL1-Blue transformed with pBluescriptIISK (), and XL1-Blue carrying the pBluescriptIISK () plasmid with wild-type recA (WT), respectively. Overnight cul-
1104 tures were diluted 1:100 in LB-ampicillin and incubated at 37 C until the culture reached an A600 of 0.7. At this reading, 1.5 ml of the culture was pelleted, resuspended in 100 ml of distilled water and stored at ÿ70 C. Serum containing polyclonal antibodies against RecA (rabbit anti-RecA) was prepared by Hazelton Research Products. Western blots were performed as described (Sambrook et al., 1989) using PVDF membranes (NOVEX). The secondary antibody was linked to alkaline phosphatase, which was detected and quantitated using the Vistra ECF Western blotting system (Amersham) and the STORM imaging system (Molecular Dynamics). ELISAs were performed in Costar's 96-well plates using 2.5 mg of total protein per well and each plate was set up in triplicate. The color change was measured photometrically at 405 nm with a Titertek Multiscan PLUS MKII reader. After background subtraction, the values obtained in triplicate were averaged and used to determine the mean and standard deviation of each mutant relative to the control lysate from XL1-Blue carrying pBluescriptIISK (), expressing wildtype recA. Measurement of RecA activity in vivo Each mutant RecA in this study was tested in three in vivo assays for its ability to carry out both homologous recombination and recombinational repair of damaged DNA. Pro®ciency in homologous recombination was assessed using a l plaque assay. The ability of the redÿgamÿ l phage, lRX (S. Pati and R.D. C.-O., unpublished results), to support plaque formation on bacterial lawns is entirely dependent on host RecA function, and we determined the ef®ciency of plaque formation on lawns of recA mutants (Enquist & Skalka, 1973). Each E. coli XL1-Blue (recAÿ) subclone harboring the plasmidborne mutant RecA was grown from a single colony on selective media in 5 ml TB broth at 30 C overnight. The culture was pelleted, then resuspended in 10 mM MgSO4 to A600 0.5. A 200 ml sample of the cells were then infected with 500 pfu of phage lRX. After 15 minutes adsorption at 37 C, 2.5 ml of TB top agar was added to the infected cells, plated onto TB plates and incubated at 37 C overnight for the growth of the bacterial lawn and viral plaque formation. Positive and negative controls included XL1-Blue-carrying wild-type RecA in pBluescriptIISK() and XL1-Blue cells transformed with pBluescriptIISK(), respectively. The number of plaques for the recA mutants was scored in comparison with the positive control. The recombinational repair activity of the plasmidborne recA mutants was tested by observing cell survival following exposure to UV irradiation and mitomycin C. For both assays a cell culture of each mutant was grown to A600 0.5 and consequently diluted with culture media to A600 0.05. From these dilutions, 3ml each were spotted onto LB agar plates containing ampicillin (100 mg/ml) and on plates containing ampicillin (100 mg/ml) and mitomycin C for the UV resistance and varying concentrations of mitomycin C (0, 0.1, 0.2, 0.3 mg/ml) for the UV and mitomycin C resistance assays, respectively. Plates were then exposed to different dosages of short wave UV light (0, 0.2, 0.4, 0.8, 1.2, 1.8 and 3.0 mJ) using the ``energy'' mode of a Stratalinker 1800 equipped with a single 8W germicidal lamp. For comparison, the dose of 3 mJ is equivalent to irradiation by a lamp producing 600 mW/cm2 intensity of UV light from a distance of 17 cm for 11 seconds.
Saturation Mutagenesis of the RecA DNA Pairing Domain After incubation at 37 C overnight, the culture spots were evaluated in comparison with the positive and negative controls. The results were interpreted as recA wild type activity, recA/ÿ less active than wild type, recAÿ/ less active than recA/ÿ but more active than recAÿ, and recAÿ inactive.
Acknowledgments We thank Peggy Hsieh, Susan Gottesman, Kyoishi Mizuuchi and Rick Proia for reading the manuscript and helpful comments. We thank George Poy, Amit Rahman and Linda Robinson for their technical help.
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Edited by M. Gottesman (Received 17 August 1998; received in revised form 27 November 1998; accepted 14 December 1998)