Analysis of recA mutants with altered SOS functions

DNA Repair

ELSEVIER

Mutation Research 336 (1995) 39-48

Analysis of recA mutants with altered SOS functions Don G. Ennis

a~*, Arthur

S. Levine a, Walter H. Koch b, Roger Woodgate a

a Section on DNA Replication,Repair and Mutagenesis,NationalInstituteof Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2725, USA b Molecular Biology Branch, Food and Drug Administration, Washington, DC 20204, USA

Received 11 February 1994; revision received 25 July 1994; accepted 25 July 1994

Abstract

The Escherichia coli RecA protein has at least three roles in SOS mutagenesis: (1) derepression of the SOS regulon by mediating LexA cleavage; (2) activation of the UmuD mutagenesis protein by mediating its cleavage; and (3) targeting the Umu-like mutagenesis proteins to DNA. Using a combined approach of molecular and physiological assays, it is now possible to determine which of the three defined steps has been altered in any recA mutant. In this study, we have focussed on the ability of six particular recA mutants (recA85, recA430, recA432, recA433, recA435 and recA730) to perform these functions. Phenotypically, recA85 and recA730 were similar in that in &A+ and le_&(Def) backgrounds, they exhibited constitutive coprotease activity towards the UmuD mutagenesis protein. Somewhat surprisingly, in a lexA(Ind-) background, UmuD cleavage was damage inducible, suggesting that the repressed level of the RecA* protein cannot spontaneously achieve a fully activated state. Although isolated in separate laboratories, the nucleotide sequence of the recA85 and recA730 mutants revealed that they were identical, with both alleles possessing a GAUGE --) Lys change in the mutant protein. The recA430, recA433 and recA435 mutants were found to be defective for both h mutagenesis and UmuD cleavage. A mutagenesis was fully restored, however, to the recA433 and recA435 strains by a low copy plasmid expressing the mutagenically active UmuD’ protein. In contrast, A mutagenesis was only partially restored to a recA430 strain by a high copy UmuD’ plasmid, suggesting that RecA430 may also be additionally defective in targeting the Umu proteins to DNA. Sequence analysis of the recA433 and recA435 alleles revealed identical substitutions resulting in Arg’43 + His. The recA432 mutation had a complex phenotype in that its coprotease activity towards UmuD depended upon the 1exA background: inducible in le.&+ strains, inefficient in lenA(Ind-1 cells and constitutive in a lexA(Def) background. The recA432 mutant was found to carry a Pro”’ + Ser substitution, a residue believed to be at the RecA subunit interface; thus this complex phenotype may result from alterations in the assembly of RecA multimers. Keywords: Escherichia coli; SOS mutagenesis;

* Corresponding

Chemiluminescent

author. Tel. (301) 496-6175; Fax (301) 402-0105.

0921-8777/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0921-8777(94)00045-X

immunoassay;

umuDC; UmuD clearlage

D.G. Ennls et al. /Mutation Research 336 (1995) 39-48

40

1. Introduction Inducible mutagenesis in Escherichia coli occurs as part of the cell’s SOS response to DNA damaging agents (Walker, 1984; Echols and Goodman, 1990). The multifunctional RecA protein plays a pivotal role in this process. Genetic analyses of numerous rec_4 mutants has identified multiple steps in the mutagenic process which require the participation of RecA protein (Mount, 1977; Blanc0 et al., 1982; Ennis et al., 1985; Dutreix et al., 1989; Sweasy et al., 1990); first, derepression of the SOS genes (e.g., urn&C) through RecA-mediated cleavage of the LexA repressor (Little and Mount, 1982); second, cleavage of UmuD protein to the mutagenically active form, UmuD’ (Shinagawa et al., 1988; Nohmi et al., 1988; Burckhardt et al., 1988); and third, a genetically defined direct role (Nohmi et al., 1988; Dutreix et al., 1989; Sweasy et al., 1990; Bailone

Table 1 Bacterial

strains

et al., 1991). This ‘thud role’ may be to interact with, and correctly position, the Umu proteins at lesions in DNA (Sweasy et al., 1990; Dutreix et al., 1992; Frank et al., 1993). Using a combination of approaches, it is now possible to distinguish which of the aforementioned steps has been altered in any recA mutant. In this study, we have focussed on the ability of six particular recA mutants (recA85, ret-4430, recA432, recA433, recA435 and recA730) because they have previously been shown to exhibit altered SOS phenotypes. recA85 is a nitrosoguanide induced mutation that was originally isolated in the Mount laboratory and has been shown to be constitutively activated for all of RecA’s functions without the normal requirement for damage (Peterson et al., 1988; Ennis, Ossana and Mount, unpublished observations). The recA430 mutation was isolated by Devoret’s lab in 1977 (Morand et al., 1977) and has previously been

used in this study

Strain

Relevant

DE1500 DE1185 DE190 DE192 DE357 DE406 DE689 DE274 DM2569 DM2571 DM2572 DM2573 DE272 DE858 DE866 DE868 DE862 DE2856 DE2857 DE2858

recA + 1exA + supD43 recA432 le.xA+ supD43 recA + lexA51 sup043 recA ’ led51 recA+ lexA3 supD43 recA+ lexA.51 recA85 lexA51 supD43 recA 730 lexA51 ArecA306 led51 supD43 ArecA306 lexA51 recA430 lexA51 supD43 recA430 lexA5l recA730 lexA51 recA432 lexA51 recA433 lexA51 recA433 lexA51 supD43 recA435 lexA51 recA730 lexA3 supD43/pRWl32, recA432 lexA3 supD43/pRW132. recA+ lexA3 supD43/pRWl32,

genotype

Source or reference

’

pDE-FL54 pDE-FL54 pDE-FL54

Woodgate and Ennis, Ennis et al., 1989 Ennis et al., 1985 Ennis et al., 1985 Woodgate and Ennis. Ennis et al., 1987 This paper Ennis et al.. 1985 Ennis et al., 1985 Ennis et al., 1985 Ennis et al., 1985 Ennis et al., 1985 This paper Ennis et al., 1988 Ennis et al., 1988 Ennis et al., 1988 Ennis et al.. 1988 This paper This paper This paper

1991

1991

a All of the strains carry thi-1, A(lac-gpt)5. suL1212, mtl-I. ilu(Ts), rpsl31. Strains DE406, DE2856. and DE2857 also carry malB :: Tn9. Strains DE274, DE689, DM2572, DM2573, DE2856 carry srlC300 :: Tn 10. DE357 carries malB45 and zja5OS :: Tn 10. ArecA306 indicates that these strains carry A(srlR-wcA)306 ::TnlO. Strains DE2856 and DE2857 were constructed from DE272 and DE858 (Ennis et al., 1989) respectively by cotransducing le.&jl(Ind-) and malB ::Tn9 using phage PI c’ir, followed by introduction of the appropriate plasmids. DE2858 was constructed from DE357 by introduction of the appropriate plasmids. pDE-FL54 (F’ laclq’ lacpL8 lacZ4504 ::Tn5) (Ennis et al., 1988) was introduced by conjugal transfer and then pRW132 was transformed into these strains.

D.G. Ennis et al. /Mutation Research 336 (1995) 39-48

shown to be defective in promoting RecA’s second role, cleavage of the UmuD protein (Burckhardt et al., 1988; Shinagawa et al, 1988). The recA432, recA433, recA435 mutations were all isolated by Kato and Shinoura in 1977 in a screen for strains that failed to promote cellular mutagenesis (Kato and Shinoura, 1977). Recent studies have, however, shown that unlike recA433 and recA435, recA432 mutants can in fact promote cellular mutagenesis functions if the strain also carries a sulA mutation (Ennis et al., 19931. The recA730 mutation was originally isolated by Witkin’s group in 1982 as a stable segregant of another recA allele, recA441 (Witkin et al., 1982). Studies have shown that in addition to promoting constitutive cellular and phage mutagenesis, red730 strains also express constitutive coprotease activity towards UmuD (Shinagawa et al., 1988; Woodgate and Ennis, 1991). The data presented here suggest that like the recA730 allele, the phenotypes of many of the other mutants also correlate with the particular RecA’s ability to promote UmuD cleavage. Furthermore, we conclude that in addition to inefficient LexA cleavage (Rebollo et al., 1984; Ennis et al., 1989) and a complete lack of UmuD cleavage (Shinagawa et al., 1988; Burckhardt et al., 19881, the RecA430 protein also has a reduced capacity in promoting RecA’s direct role in mutagenesis: targeting of the Umu proteins to DNA. DNA sequence analysis of the previously uncharacterized mutants (recA85, recA432, recA433, recA435 and recA730) and comparison of their changes to the proposed three-dimensional structure of the RecA protein (Story et al., 1992) further helps explain the observed phenotypes. 2. Materials and methods Bacterial strains

Bacterial strains used in this study are listed in Table 1 and were constructed by standard methods of conjugation, Pl transduction and plasmid transformation (Miller, 1972). Plasmid construction

DNA was isolated and manipulated by standard techniques (Sambrook et al., 1989). pRW66 is

41

a low copy plasmid carrying umuD’ (Woodgate et al., 1994). pRW134, a low copy plasmid carrying the umuD’C operon, was constructed by ligating a - 2-kb DraI fragment from pGW2123 (Nohmi et al., 1988) with XhoI linkers into the Sal1 site of pGB2 (Churchward et al., 1984). pRW132 is a medium copy plasmid that expresses the umuD gene under the control of the lac operator-promoter regulatory region: it was constructed from pSE192 (Ennis et al., 1988) (which expresses both the UmuD and UmuC proteins) by introducing a frameshift mutation at the unique MluI site N 24 nucleotides from the start of the umuC gene. A Mutagenesis assays SOS mutagenesis was measured by a A L(amI63 + AL + reversion assay that has been described previously (Ennis et al., 1985; Hauser et al., 1992). Briefly, bacterial strains were grown at 37°C to a density of approximately 3 x 10’ cells/ml, harvested and resuspended at 7 x 10’ in 10 mM MgSO, and then infected with irradiated ADE57 (ALcam ~1857). ADE57 phage stocks were diluted lo-fold into SM diluent and irradiated at 160 J/m2, yielding a survival of approximately 3% on uninduced wild-type hosts (DE1500). Where indicated, mitomycin C (Sigma) was added to a final concentration of 1 kg/ml 2 h prior to cell harvesting. When the survival of the mitomycin C treated host was too low to support plaque development, additional untreated cells of the same genotype were added after absorption to allow for phage growth. After overnight incubation at 37°C SUS+ revertants were scored on a sup+ host and total progeny scored on a supD43 strain with an otherwise identical genotype. The frequency was determined as the titer of the revertants divided by the total viable progeny. Chemiluminescent immunoassays

Polyclonal antibodies raised to highly purified proteins have been described previously (Woodgate et al., 1989; Woodgate and Ennis, 19911, and were a gift from the late H. Echols (University of California, Berkeley). These antibodies are highly specific, only cross-reacting with two or three other proteins in whole-cell E. E. coli UmuD/D’

42

D.G. Ennis et al. /Mutation Research 336 (1995) 39-48

coli extracts,

and they recognize UmuD and UmuD’ equally well (Woodgate and Ennis, 1991). The UmuD/ D’ proteins were detected in whole-cell E. coli extracts using the Western-light chemiluminescent assay (Tropix, MA) essentially as described previously (Woodgate and Ennis, 1991; Hauser et al., 1992). Like the A mutagenesis assays, where noted, cells were incubated with mitomycin C (1 pg/ml) 2 h prior to cell harvesting to activate RecA protein. Whole cell extracts from - 1 x lo* cells were separated by electrophoresis in a 15% acrylamide-SDS gel. Proteins were transferred to a nitrocellulose support membrane and incubated with a 1: 20000 dilution of the primary antibody. After appropriate washes, the membrane was incubated with a 1: 3000 dilution of a goat anti-rabbit antibody conjugated with alkaline phosphatase (Bio-Rad). Proteins were visualized by incubating the membrane with chemiluminescent substrate and exposing the membrane to X-ray film for an appropriate period of time. The relative level of the UmuD and UmuD’ proteins was obtained by densitometric analysis of films. The levels were usually estimated from multiple film exposures so that the desired bands were in the linear range of the film (Woodgate and Ennis, 1991).

DNA sequence analysis of recA mutants A - 1274-bp region of the E. co/i chromo-

some encompassing the entire recA gene was amplified via the polymerase chain reaction (Saiki et al., 1988) using two synthetic oligonucleotides, (5’-GCATTGCAGACCTTGTGGC-3’ and 5’CGACGGGATGTTGATICTG-3’). Once amplified, the double-stranded PCR product obtained from strains DE689 (recA8_5), DE274 (recA730), DE858 (recA432), DE866 (recA433) and DES62 (recA435) was asymmetrically re-amplified to generate single-stranded DNA (Gyllensten and Erlich, 1988). The DNA sequence of each mutant was obtained using the dideoxy chain terminating method (Sanger et al., 1977) with synthetic oligonucleotide primers spaced about -200 bp apart. Asymmetric DNA amplification and DNA sequencing were performed by Lark Sequencing Technologies, Inc. (Houston, TX).

3. Results DNA sequence changes of recA mutants

To gain better insight into the structure-function relationship of these six recA alleles, we have determined the nucleotide changes in the recA85, recA730, recA432, recA433 and recA435 mutants (Table 2). As noted in the Introduction, the recA730 mutation was obtained as a segregant of the recA441 allele. recA441 has two mutations in the recA gene (Knight et al., 1984; Kawashima et al., 1984) that result in G1u38 + Lys and Ile298 --f Val changes in the protein. It has long been assumed (but never proven) that recA730 contained only the former mutation. Analysis of the recA730 gene indeed confirmed the expected mutational change. Phenotypically, recA8.5 and recA730 are very similar (Peterson and Mount, 1987; Ennis et al., 1989; Ennis, Ossana and Mount, unpublished observations). Sequence analysis revealed that like recA730, recA85 has an identical change in recA that results in a substitution of Glu”’ + Lys. Interestingly, Wang and Tessman (19861 have also identified the very same change in their recA* mutant, recA1211, suggesting that this GC + AT substitution is at a mutational ‘hot spot’. Although phenotypic analysis was performed on both recA85 and recA730 alleles, for the sake of simplicity, only the results with recA730 are presented. The recA433 and recA435 alleles also exhibited similar phenotypic characteristics to each other. Nucleotide sequence analysis of both of these genes revealed that they too were identical

Table 2 Nucleotide and amino acid changes in recA mutants recA allele

Nucleotide change a

Amino acid change h

recA 730 recA85 recA433 recA435 recA432

lh4G --f A lh4G -+ A “‘G + A “‘G + A ‘“‘C + T

G1u3*+ Lys GIu3* --f Lys Arg243 + His Arg243 -+ His Pro”” + Ser

a Numbering system of Horii et al. (1980), based upon the start of the recA transcript. ’ Numbering based upon the residue in the mature RecA protein.

D. G. Ennis et al. /Mutation Research 336 (1995) 39-48

(Table 2). In this case, the changed nucleotides caused a substitution of Arg243 -+ His. Again, phenotypic studies were performed on both recA433 and recA43.5 alleles, but only the data obtained with recA433 are shown for simplicity. Interestingly, Devoret and colleagues (Dutreix et al., 1989) reported a recA mutant, recA1734, that has similar characteristics to recA433 /435. Their recA1734 mutant also causes a change at the same residue but results in an Arg243 -+ Leu substitution. The recA432 allele was found to be novel in that it results in a Pro”” -+ Ser substitution. Effects of le.xA mutations on the coprotease activity of RecA432 and RecA730 proteins recA432 and recA730 strains share certain

phenotypic characteristics in that they have previously been shown to behave as recA* mutants and exhibit coprotease activity towards AC1 and UmuD without the normal requirement for inducing treatments in a ZexA(Def) background (Peterson et al., 1988; Ennis et al., 1989). They differ, however, in that while these activities are still constitutive in recA730 lexA + strains, A mutagenesis and LexA cleavage in recA432 lexA + strains are inducible functions (Ennis et al., 1989, 1993). Cleavage of UmuD appears to parallel these observations. For example, in a recA432 fexA+ background, UmuD was induced slightly in the absence of damage, and upon exposure to mitomycin C, was processed with similar efficienIexA 3 recA+ recA 432 recA 730 -+ -+ -+

43

ties as the recA+ lexA+ control, with approximately 50% of induced UmuD converted to UmuD’ (Fig. 11. However, the recA432 l&(Def) strain exhibited constitutive coprotease activity that was indistinguishable from the recA730 strain (Fig. 1). To examine this differential activation more accurately, UmuD cleavage was compared under conditions where the RecA* proteins might be expected to be limiting, i.e., in a lexA3(Ind-1 strain. The LexA3(Ind-1 repressor is known to be resistant to cleavage (Little and Mount, 1982; Lin and Little, 19881, and thus the mutant RecA proteins would be present at basal levels in this background. Since the level of chromosomal UmuD expression is also barely detectable in the lexA3(Ind-) background (Woodgate and Ennis, 19911, UmuD was expressed from a plasmid (pRW132) which placed the umuD gene under the control of the lac operator-promoter region. Without IPTG inducing treatment, this transcriptional fusion produces a similar level of UmuD to that expressed from the chromosomal umuD gene in a lexA(Def) strain (Woodgate and Ennis, 1991). In a previous study (Woodgate and Ennis, 19911, we suggested that derepressed levels of activated RecA protein were required for efficient UmuD cleavage. This suggestion was recently challenged by Sommer et al. (19931, who noted that UmuD cleavage occurred if the recA+ fexA(Ind-) strains were exposed to DNA damaging agents. Our present studies agree with Sommer et al., and we have extended these observations further by not-

IexA + recA + recA432 -+ -+

/exA recA’ -+

(Def)

recA432 recA 730 -+ -+

Fig. 1. Effects of a le.1~4 mutation on UmuD cleavage in recA+. recA432 and recA730 strains. The UmuD and UmuD’ protems were detected in extracts from various E. coli mutants using the chemiluminescent immunoassay. As noted in Materials and methods, in the lexA(Ind-) strain, UmuD was expressed from uninduced pRW132, while in the [exA+ and leul(Def) strains, UmuD was expressed chromosomally. Whole-cell extracts were obtained from DE2856, DE2857. DE285S. DE1500. DE1185, DE406, DE858, and DE274. Cells were either uninduced ( -) or treated with mitomycin C (1 Kg/ml) (+ 1 for 2 h prior to cell harvesting to activate RecA functions. The recA allele of the strain is indicated above its appropriate track. The positions of UmuD and UmuD’ are indicated by arrows on the left and right of the figure.

D.G. Ennis et al. /Mutation Research 336 (1995) 39-48

44

ing that like the recA+ strain, the recA730 strain also promoted UmuD cleavage after DNA damage (Fig. 1). recA* mutants, like recA730, are thought to be activated because they have a relaxed specificity for activating cofactors and thus bind to normal cellular components or ‘endogenous signals’ (Wang et al., 1988). Our finding that the RecA730 protein is activated by DNA damage implies that the endogenous signals may also be limiting in the repressed lexA3(Ind-) mutant strains. In comparison, UmuD cleavage in the recA432 lex_43(Ind-) strain was even less efficient than that observed in the recA+ lexA3(Ind-) control (Fig. 1). These results are therefore consistent with other studies, leading to the conclusion that the RecA432 mutant protein is deficient in obtaining an activated state when present at basal levels (Ennis et al., 1993). RecA433 is selectively defective in coprotease activity towards UmuD whilst RecA430 has additional defects Mutants carrying alleles such as ArecA306, recA430, and recA433 are unable to support

phage mutagenesis (Ennis et al., 1985, 1989). As shown in Fig. 2, each of these mutants was also defective for UmuD cleavage even after exposure to 1 pg/ml mitomycin C. In contrast, the recA433 allele is known to be highly proficient for the induction of SOS genes and h prophage, indicat-

lexA (Def)

recA+ -+

recAA -+

recA430 -+

recA433 -+

+UmuD

ale

+lJmuD’

Fig. 2. UmuD cleavage in recA mutants defective for mutagenesis. Chromosomally encoded UmuD(D’) proteins were detected in extracts from various E. coli recA k.wKDef) mutants as described in Materials and methods. Extracts were obtained either from uninduced DE406, DM2569, DM2572, and DE866 ( - ), or from cells treated with mitomycin C (1 kg/ml) (t ) to activate RecA functions.

ing that it has retained the coprotease activity towards LexA and AC1 repressors (Ennis et al., 1989). Recently, we have demonstrated that recA433 can also efficiently cleave the MucA mutagenesis protein, thereby restoring A mutagenesis to the mutants (Hauser et al., 1992). It would appear therefore that the RecA433 protein is selectively defective for UmuD cleavage. In contrast, RecA430 protein appears to have a generally reduced ability to mediate posttranslational cleavage. Both LexA and MucA cleavage are reduced compared to wild-type RecA (Rebollo et al., 1984; Ennis et al., 1989; Hauser et al., 1992) and no cleavage is observed with the AC1 repressor (Roberts and Roberts, 1981) or UmuD proteins (Shinagawa et al., 1988; Burckhardt et al., 1988). Previous studies have demonstrated that cellular mutagenesis can be restored to certain recA mutants defective in UmuD cleavage by introducing a plasmid that expresses UmuD’ into the strain (Nohmi et al., 1988; Dutreix et al., 1989; Sweasy et al., 1990). A notable exception is the ArecA mutation which remains non-mutable despite the presence of UmuD’ (Nohmi et al., 1988; Sweasy et al., 1990). Detection of cellular mutagenic events in the hypersensitive ArecA strain is difficult, however, because the UV fluences sufficient to induce mutations are likely to be lethal. This difficulty is reduced by use of the phage assay, since plating efficiencies and detection of mutant phage are not severely impaired even when using an irradiated host that is hypersensitive (Ennis et al., 1989; Hauser et al., 1992). In agreement with the cellular mutagenesis experiments, we found that expression of UmuD’ was unable to restore phage mutagenesis to a ArecA lexA(Def) strain (Table 2). In contrast, introduction of high or low copy UmuD’ plasmids (pGW2122 and pRW66 respectively) into the recA433 strain fully restored A mutability (Table 2). Introduction of pRW66 into the recA430 strain, however, resulted in a modest restoration of phage mutagenesis; only a 2-3-fold increase over the same strain lacking UmuD’ (Table 2). Expression of both UmuD’ C proteins from a related low copy plasmid (pRW134) resulted in a modest stimulation of mutagenesis for the

D.G. Ennis et al. /Mutation Research 336 (199.~) 39-48

recA430

mutant, but it was still substantially less than that observed in the recA433 strain with plasmids encoding UmuD’. h mutagenesis was partially restored to the recA430 strain by a high copy UmuD’ plasmid (pGW2122) but only to approximately half of that observed in the recA+ control or the recA433 mutant strain (Table 2). Expressing UmuD’ from either the high (pGW 2122) or low (pRW66) copy plasmid yielded an equivalent level of mutagenesis in the latter strains (Table 2), suggesting that the UmuD’ expression from the lower copy plasmid was already saturating for mutagenesis. Indeed, the same high levels were observed in the recA + and recA433 strains even when UmuD’ was produced from a single copy episome (unpublished observations). The observation that phage mutagenesis in the recA430 strain was inefficient, especially when UmuD’ was expressed from the lower copy plasmid, implies that the RecA430 protein is partially defective in promoting RecA’s ‘third role’ and that overproduction of UmuD’ from the high copy plasmid can partially compensate for this defect. Conversely it would appear that the RecA433 protein is highly proficient in promoting the ‘third role’.

4. Discussion Structure-function relationships of the recA mutants The recA433 mutant is defective for UmuD

cleavage, but proficient at cleavage of all other target molecules tested. Additionally, it was able to efficiently promote RecA’s third role in SOS mutagenesis. In this respect, recA433 mimics recAZ734 which is also selectively defective for UmuD cleavage, and performs the third role (Dutreix et al., 1989). Surprisingly, this phenotype appears to be caused by changes in the very same Arg 243 residue. recA433 has an ArgZ4” + His change, while recA1734 has Arg24’ + Leu (Dutreix et al., 1989). Both crystallographic and electron microscopic studies (Story et al., 1992; Yu and Egelman, 1993) suggest that this residue is in close proximity to LexA and UmuD when they bind to RecA’s deep helical groove. It would

45

appear that the substitutions of His and Leu at residue 243 specifically abolish coprotease activity towards UmuD without affecting similar activities toward LexA, CI or MucA. recA730 belongs to a class of recA* mutants that is thought to be constitutive for all known RecA functions including UmuD cleavage. Our studies here suggest that when expressed at repressed levels, at least RecA730 has a reduced ability to become spontaneously activated for UmuD cleavage. The recA730 mutation occurs in the amino portion of the protein, and this mutation results in the efficient formation of nucleoprotein filaments (Story et al., 1992; Laverly and Kowalczykowski, 1992). It is interesting to note that the very same mutations were isolated in three separate laboratories, implying a mutagenic ‘hot spot’. Clearly, changes at this position are critically important for RecA’s ability to spontaneously form nucleoprotein filaments. The recA430 allele appears to be partially or completely defective for the various RecA functions examined here. In addition, recA430 mutants are known to be partially defective in promoting homologous recombination (Morand et al., 1977). In contrast however, RecA430 can promote cleavage of the 480 CI repressor protein efficiently (Devoret et al., 1983; Eguchi et al., 1988). The mutation in recA430 causes a change of Gly to Ser at amino acid residue 204 (Kawashima et al., 1984). This mutation resides in a stretch of residues called L2, a disordered region in the RecA crystal structure that has been hypothesized to be involved in DNA binding (Story et al., 1992). Indeed, some of these phenotypes (LexA/ UmuD cleavage and recombinational inefficiency) can be attributed to RecA430’s inability to bind efficiently to DNA and form nucleoprotein filaments (Lu and Echols, 1987; Menetski and Kowalczykowski, 1990). This does not, however, completely explain the partial defect of RecA430 in promoting the third role. For example, both A and cellular mutagenesis can be fully restored to recA430 mutants by expressing functional homologs of UmuDC such as MucAB, from either low or medium copy plasmids (Blanc0 and Rebollo, 1981; Blanc0 et al., 1986; Hauser et al., 1992). These findings, together with our ob-

46

D.G. Ennis et al. /Mutation

Table 3 A mutagenesis assays Genotype ~ recA

IexA

A306

51

+

51

430

51

433

51

MC

+ + + +

Reversion frequency (Sus+ revertants per 10’ survivors) No plasmid

pGW2122 (pUC-D’)

pGB2 (pSCvector)

pRW66 (pSC-D’)

4 10 15 330 6 14 4 9

10 9 550 620 320 310 610 630

nd a nd 17 310 nd nd 8 3

4 11 600 590 35 h 40 h 650 700

See Materials and methods for all experimental procedures. All data are the average of two or three independent experiments. The ArecA306 lexA51 mutant strains used were DM2569 and DM2571. The recA+ lexA51 strains were DE190 and DE192. The recA430 lexA51 strains were DM2572 and DM2573, and the recA433 lexA51 strains were DES66 and DE868. For full genotypes of each strain see Table 1. a nd, not determined. ’ Mutagenesis increased to 109 (-MC) and 95 (+ MC) revertants per 10’ survivors upon introduction of a low copy plasmid (pRW134) encoding the UmuD’ and UmuC proteins.

servation that a low copy UmuD’ plasmid does not restore A mutagenesis, suggest that the mutagenesis phenotype results from RecA430’s reduced ability to interact with the chromosomally encoded UmuDC proteins but not the plasmidencoded MucAB proteins. In this respect, RecA430 is similar to another mutant, RecA1730, which interacts poorly with the Umu proteins but efficiently with the MucAB proteins (Bailone et al., 1991; Frank et al., 1993). In contrast to the small increase with the A assay (Table 3), cellular mutagenesis was partially restored to a recA430 mutant by a low copy UmuD’ plasmid (pRW66) but somewhat less than that observed in the recA+ control (Nohmi et al., 1988; Sweasy et al., 1990). Furthermore, introduction of a low copy plasmid encoding both UmuD’ and UmuC proteins yielded a higher level of cellular mutagenesis than the UmuD’ plasmid alone (Sweasy et al., 19901, suggesting that not only is UmuD’ limiting for recA430 mutant strains but so is UmuC. We suspect that the differences observed between cellular and A phage mutagen-

Research 336 (1995) 39-48

esis may reflect the apparent partial defect of RecA430 in promoting the ‘direct role’, Replication of the smaller h replicon is much faster than that of the bacterial chromosome, undergoing many rounds of replication in the same time that the cell only replicates once (Verma et al., 1989). It is possible that in this time frame, RecA430 may be unable to successfully target the UmuD’ produced from the low copy plasmid to the phage DNA. Finally, the ~~4432 mutant is more complex but may offer insights into RecA activation. In recA432 led + cells, RecA432 becomes activated by the signals produced by DNA damage and induces the SOS response. Once induced and LexA is cleaved, RecA432 builds up to high derepressed levels and its activation apparently leads to chronic activation and persistent derepression of SOS genes (Ennis et al., 1993). We show here that UmuD cleavage is also inducible in recA432 leaA + cells but becomes constitutive in recA432 lexA(Def) strains (Fig. 1). These cleavage patterns for UmuD confirm our previous interpretations for RecA432 activation, based on LexA cleavage studies (Ennis et al., 1993); first, at low repressed levels, RecA432 is not activated, requiring damage-induced signals for activation, and second, at high protein levels endogenous signals are apparently sufficient for activation. We demonstrated that in recA432 fexA(Ind-) mutants, UmuD cleavage was less efficient following a mitomycin C treatment than the recA+ lexA(Ind-) control (Fig. l), indicating that when RecA432 is fixed at the low repressed level, activation by damage-induced signals is also inefficient. To explain these mutant properties, we propose that there may be a defect in the assembly of RecA432 multimers and that increasing the level of the mutant protein not only compensates for the defect but spontaneously forms activated complexes. The nucleotide change in recA432 caused a substitution of Pro”’ -+ Ser and to our knowledge is the first mutation at this position. Pro”’ is in a loop on the protein surface (residues 118-123), which was implicated as part of a subunit interface (Story et al., 1992). An opposing loop that forms the interface on the adjacent monomer has also been identified (positions 210-

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D.G. Ennb et al. /Mutation Research 336 (1995) 39-48

222); structural studies and mutational analyses of this opposing loop have identified several key residues which are likely involved in interface contacts with the neighboring monomer (Skiba and Knight, 1994). Crystallographic studies of RecA filaments revealed that two of these critical interface amino acids (Lys216 and Phe*“) are in close proximity to Pro”’ on the neighboring monomer (Story et al., 1992; Skiba and Knight, 1994). A substitution for a proline residue might be expected to increase the flexibility of this loop, altering multimeric assembly and thus accounting for the abnormal activation properties of RecA432. Comparisons of the primary structures of approximately 60 bacterial RecA proteins have revealed that Pro”’ is an invariant amino acid (Rota and Cox, personal communication); this conservation implies that this residue is functionally important, presumably in the formation of multimers. Acknowledgements

We are grateful to the late Hatch Echols for the gift of UmuD/D’ antibodies. We would like to thank Graham Walker for plasmids pGW2122 and pSE192; Kendall Knight, Ekaterina Frank and Janet Hauser for helpful discussions; and AIberto Rota and Michael Cox for sharing unpublished information. References Bailone, A., S. Sommer, J. Knezevic, M. Dutreix and R. Devoret (1991) A RecA protein mutant deficient in its interaction with the UmuDC complex, Biochimie, 73, 479484. Blanco, M. and J.E. Rebollo (1981) Plasmid pKMlOl-dependent repair and mutagenesis in Escherichia coli cells with mutations lenB30, tif and zab-53 in the recA gene, Mutation Res., 81, 265-275. Blanco, M., G. Herrera, P. Collado, J.E. Rebollo and L.M. Botella (1982) Influence of RecA protein on induced mutagenesis, Biochimie, 64, 633-636. Blanco, M., G. Herrera and V. Aleixandre (1986) Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis of Escherichia coli, Mol. Gen. Genet.. 205, 234-239. Burckhardt, SE., R. Woodgate, R.H. Scheuermann and H. Echols (1988) UmuD mutagenesis protein of Escherichia

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