Biochimie ( 1991 ) 73, 281-284 © Socirt6 franqaise de biochimie et biologie moirculaire / Elsevier. Paris
,'oi
Site-directed m u t a g e n e s i s ir,l the Escherichia coil recA gene C Cazaux, F Larminat, M Defais* Laboratoire de Pharmacologie et de To.ricologie Fondamentales. CNRS, 205 route de Narbonne. 31077 Toulouse Cedex. France
(Received 19 October 1990: accepted 10 December 1990)
Summary - - Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its i~volvement in an ATP binding site, as previously shown in vitro [ 1|. RecA / recombination / SOS repair
Introduction
Materials and M e t h o d s
The first discovered role o f E coli R e c A protein was its r e c o m b i n a s e activity [2, 3]. However, R e c A has another p r i m a r y function in the control of the SOS s y s t e m [4-6] in which it acts as a protease cofactor 17! a l l o w i n g the cleavage o f L e x A repressor [81. Moreover, R e c A participates in the expression o f m a n y SOS functions such as post-replicative r e c o m b i n a t i o n [9, 10], repair o f double-strand breaks [1 1] or induced m u t a g e n e s i s [ 1 2 - 1 7 ] . To p e r f o r m these f u n c t i o n s , R e c A has to be activated b y interaction with singlestrand D N A and A T P [18]. These multiple functions o f R e c A protein suggest the presence o f several active sites in the protein w h i c h have been located on the r e c A gene according to the different mutant phenotypes [ 1 9 - 2 2 ] . P r e v i o u s genetic studies led to the c o n c l u s i o n that R e c A possessed different d o m a i n s for r e c o m b i n a t i o n a n d c o p r o t e a s e a c t i v i t y [23, 24]. R e c e n t l y it has been s h o w n that these d o m a i n s m a y overlap [22; O g a w a et al, u n p u b l i s h e d results]. In this study we attempted to define in the R e c A protein a d o m a i n o f interaction with either the L e x A repressor or with ATP, using specifically constructed mutations o f the r e c A gene, whose p h e n o t y p e s we s u b s e q u e n t l y e x a m i n e d in vivo.
Media and growth conditions
*Correspondence and reprints
Cultures were grown in Luria broth (Difco) at 37°C. Plasmidharbouring cells were always grown in the presence of I U U ~..L~IIIII dlll[.311,.,llltll ~,O1~111~1.]. IYIIU3 ~ l i n | m a | medium su emented with 0.4% glucose. 0.5% casamino acid and 1 lag/ml thiamine [311 was used for the 13-galactosidase assay. McConkey medium (Difco) supplemented with 1% lactose was used for the recombination assay. Oligonucleotide-directed mutagenesis ofrecA gene
A 3.l-kb fragment carrying the E coli recA gene and excised by BamHl digestion from the plasmid pFL352 [25] was cloned into the BamHI site of M 13mp 10 [26]. M 13mp 10/recA phages were selected and single-stranded phage DNA were purified as described by Messing 127].The recA mutants were prepared as described by Eckstein [28]. Mutagenic primers used to produce mutations were synthetized using DNA synthetiser 38!A (Applied Biosystems). Each mutation created a restriction site for facilitating the screening of the mutated phages. The duplex DNA was used to transform competent JM101 [29] or TG! cells [30]. Individual plaques were selected and phage DNA carrying the desired mutation were identified by digestion with the appropriate restriction enzyme. SOS induction
Colicin E1 induction with cea::lacZ fusions was nleasured as previously described [31], using the strains FL8641 [25] and FL8642, recA430.
282
C Cazaux et al
Dctermimttion q~flac recombination
Table I. Amino acid changes and mutant denomination.
intrachromosomal recombination was measured in FL7023 ~GY7023 ~'ecA) bacteria harbouring a recombinant plasmid or canwino the recA4 ¢0 mutation as described [ 171.
Mutated protein
Change in nucleotide
Change in protein sequence
RecA694 RecA430 RecA604 RecA605 RecA659 RecA611 RecA664
A T T - - > GTT GGT m > AGT GGT ~ > GCG G G T - - > GTT GAA - - > CAA GGT m > GCG TAC - - > GAG
lie (199) - - > Val Gly (204) - - > Ser Gly (204) - - > Ala Gly (204) - - > Val Glu (207) - - > Gin Glu (211) ~ > Ala Tyr (264) - - > Glu
Bacterhd amt phage assays The strain used was JC!0289 I321. Phages were lambda+ 1331. UV irradiation, cell survival and lambda phage survival assays ~ere carried out as described previously 141.
Results and Discussion In order to dissociate the different active sites of RecA protein we constructed a series of point mutations in the recA gene (table I). We first changed codon 839 coding for Tyr 264. This amino acid has been shown in rin-o to interact with ATP [ 1]. The replacing amino acid was Glu, bearing a negative charge compared to tyrosine and leading to the mutation recA664. Two mutations, recA604 and recA605, were created at the site of the recA430 mutation which is known to affect the coprotease activity of the protein, ie the l i k e l y site of i n t e r a c t i o n with L e x A r e p r e s s o r . recA430 mutants in which wild-type Gly 204 is replaced by Ser show normal recombinase activity I341. RecA604 and RecA605 proteins have comparable amino acid substitution, carrying 1 or 2 additional methyl ~ o u p s which yield the amino acids Ala and Val respectively. To further a.n.a!y~e the extent of R e c A - L e x A interaction we constructed 3 mutants, one of which, recA694, is located 5 amino acids upstream of the recA430 mutation. The last 2 mutations were respectively 3 and 7 amino acids downstream from the same mutation. In these cases substitution changed neither the charge, nor the hydrophobicity, nor the steric hindrance of the amino acids, which permitted us to analyse the effect of presumably minor modifications of the protein. These mutations were cloned in pBR322 and used to transform a ArecA strain. We then analysed the capacity of these new recA mutants to induce and express the SOS functions and to ca;ry out intrachromosomal recombination. Table II summarizes the results obtained with each mutant. The recA664 mutant behaved as a recA deletion strain. Tyr 264 had been previously shown to be essential to the in vitro binding of ATP to RecA protein [1]. Hence it is conceivable that changing Tyr into GIo destroyed the ATP binding site, leading in vivo to a complete loss of ar;y RecA activity. This finding is also in agreement wi:h the crystal structure of RecA, which indicates that ADP binds to Tyr264 and to amino acids 71-73 [Story and Steitz, unpublished results].
recA604 and recA605 mutations were located at amino acid 204. In these mutants we expected a phenotype analogous to that observed for the recA430 mutant, ie slight UV sensitivity, decreased SOS induction and normal recombination capacities. It can be seen in table II that adding 1 or 2 methyl groups to amino acid 204 affects UV sensitivity only slightly. The ability of these mutants to induce the SOS response was measured using a cea::lacZ fusion containing a ' p r o m o t e r up' mutation in order to increase sensitivity [25, 35]. The kinetics of induction of B-galactosidase activity were identical in all 3 mutants recA430, recA604 and recA605. Thus it appeared that the capacity of these mutants to promote LexA self-cleavage was decreased compared to the wild-type. These mutants were also deficient in SOS repair since they were unable to reactivate UV irradiated lambda phage and no mutagenesis could be detected in phage progeny (data not shown). This . . . . '-1 be ~. h. l.,.J l'-:---~ d l l l g ~ L I by ""-LIIC '~ lgltL, lat t:uuiu . . . t. t l'-~" the mutants are unable to derepress the SOS response, and therefore the functions necessary for Weigle repair were not transcribed. Alternatively, the mutant proteins may be unable to promote UmuD cle~'vage which is required for mutagenesis [ 16]. Intrachromosomic recombination was unaffected by the presence of one methyl group on residue 204. Table IL SOS and recombination activities. Strains
UV cea::lacZ Weigle resistance induction reactivation of lambda
Recombinant activity
RecA÷ RecA430
+++ +
RecA694 ++ RecA659 . RecA611 . RecA604 RecA605
+++ _
+++ ++
++
++ . .
+++
_ _
++ _
. .
+ +
RecA664 .
+++ + . . + + .
.
.
Site-directed re~'A mutants The r e c o m b i n a t i o n e f f i c i e n c y o f r e c A 6 0 4 mutant was i d e n t i c a l to that o b s e r v e d w i t h r e c A 4 3 0 m u t a n t . H o w e v e r , the addition o f a second m e t h y l group at this position led to a strong decrease in r e c o m b i n a s e activity without reaching a recA nul phenotype. T h e s e data indicate that a m i n o acid at position 204 w h i c h at first appeared to be i n v o l v e d only in the interaction with L e x A repressor could also be required for r e c o m bination. To further study the e x t e n s i o n of' L e x A interaction around residue 204, the mutation r e c A 6 9 4 , w h i c h is located 5 a m i n o acids u p s t r e a m from residue 204 and r e c A 6 5 9 and recA611 mutations, 3 and 7 a m i n o acids d o w n s t r e a m , were analysed. R e c A 6 9 4 protein s e e m e d to be only slightly affected in the SOS response and in recombination. However, recA659 and recArli mutants lost all R e c A functions and b e h a v e d as recA deletion strains, despite the fact that they produced n o r m a l quantities of the mutated protein as detected by S D S - P A G E (data not shown).
Conclusion It thus appears that residue 204 could be at the edge o f a site w h i c h extends at least 7 a m i n o acids d o w n stream. This residue plays a key role, since a slight modification o f the a m i n o acid structure has a d r a m a t i c e f f e c t on R e c A a c t i v i t y ( r e c A r 0 5 ) . T h e u p s t r e a m region m a y not be involved in any R e c A function. The d o w n s t r e a m J egion m a y interact not o n l y with L e x A r e p r e s s o r but p r o b a b l y also with ATP ~in¢'~ m,,tnnt S c h a n g i n g "~ Aiff. . . . , ~.~,~ . . . . ~n this region lose all R e c A functions - not only repair, but also recombination. O v e r l a p p i n g of A T P b i n d i n g and L e x A interaction d o m a i n s at a m i n o acids 200 and 205 has b e e n also d e m o n s t r a t e d by O g a w a et al ( u n p u b l i s h e d results). It cannot be confirmed by the c r y s t a l l o g r a p h i c structure d o n e in the p r e s e n c e o f A D P and in the absence o f D N A (Story and Steitz, u n p u b l i s h e d results) w h i c h could create a different c o n f o r m a t i o n a l c h a n g e t h a n that p r o v o k e d in the p r e s e n c e o f A T P and D N A [36]. . . . .
,
. . . .
~ v
l l i ~ l ~ i
~
'll,l ~ l I 1 1 ~ , ~ 1 ~¢~,'I i I~
I ~1.¢ II~l 1 ' l t ~ l I'~1 ~lm~ ~ I
I11
Acknowledgments The authors acknowledge the collaboration of H Mazarguil for synthesis of oligonucleotides. This work was supported in part by the Regional Council of Midi-Pyrrnres.
References Knight KL, McEntee K (1985) Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azido adenosine 5'-triphosphate. J Biol Chem 260, 10185-10191
283
2 Clark AJ, Margulies AD (1965) Isolation and characterization of recombination-deficient mutants of Es~'heri~hia coli K 12. Proc Natl Acad Sci USA 53, 451-459 3 Cox MM, Lehman IR (1987) Enzymes of general recombination. Ann Rev Biochem 56, 229-262 4 Defais M, Fauquet P, Radman M, Errera M (1971) Ultraviolet reactivation and ultraviolet mutagenesis of lambda in different genetic systems. Virology 43, 495-503 5 Radman M (1974) Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis. In: Molecular and Environmental Aspects ofMutagenesis (Prakash L, Sherman F. Miller M, Lawrence C, Tabor HW, eds) CC Thomas, Springfield, IL, 128-142 6 Radman M (1975) SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. In: Molecular Mechanisms for repair of DNA (Hanawait P, Setlow RB, eds) Plenum Publishing Corp, NY, 355-367 7 Little JW (1984) Autodigestion of LexA and prophage repressors. Proc Natl Acad Sci USA 81, 1375-1379 8 Walker GC (1985) Inducible DNA repair systems. Ann Rev Biochem 54, 425-457 9 Rupp WD, Howard-Flanders P (1968) Discontinuities in the DNA synthesized in an excision-deficient strain of Escherichia coli following ultraviolet irradiation. J Mol Biol 31,291-304 10 Hanawalt PC, Cooper PK, Ganesan AK, Smith CA (1979) DNA repair in bacteria and mammalian cells. Ann Rer Biochem 48, 783-836 11 Brozmanowa J, lljina TP, Saenko AS, Sedliakova M (1981) Inducible repair of DNA double strand breaks in Escherichia coli. Stud Biophys 85, 195-201 12 Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40, 869-907 13 Ennis DG, Fisher B, Edminton S, Mount DW (1985) Dual role for Escherichia coli RecA protein in SOS mutagenesis. Proc Natl Acud Sci USA 82, 3325-3329 14 Shinagawa H, lwasaki H, Kato T, Nakata A (1988) RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85.1806--I 810 15 Burckardt SE, Woodgate R, Scheuermann RH, Echols H (1988) The UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc Natl Acad Sci USA 85, 1811- 1815 16 Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85, 1816-1820 17 Dutreix M, Moreau PL, Bailone A, Galibert F. Battista JR, Walker GR, Devoret R (1989) New recA mutations that dissociate the various RecA protein activities in Es~'herichia coli provide evidence for an aoditional role for RecA protein in UV-mutagenesis. J Bacterioi 2415-2423 18 Craig NL, Roberts JW (1980) Escherichia coli RecA protein-directed cleavage of phage lambda repressor requires polynucleotide. Nature 283, 26-30 19 Kawashima H, Horri T, Ogawa T, Ogawa H (19~4) Functional domains of E coli RecA protein deduced from mutational sites in the gene. Mol Gen Genet 193,288-292 20 Makino O, Shibata Y, Maeda H, Shibata T, Ando T (1985) Monoclonal antibodies with specific effects on partial activities of recA protein of E coli. J Biol Chem 260 15402-15405
284 21 22
23
24
25 26
27 28
C Cazaux et a/ Ogax~a H. Ogawa T (1986) General recombination: functions and structure of RecA protein. Adv Biophys 21, 135-148 Wang WB. Tessman ES (1986) Evidence that the recA441 (tif) mutant of Escherichit~ coli K-12 contains a thermosensitive intragenic suppressor of RecA ca,nstitutixe protease activity. J Bacteriol 168, 901-910 Morand P. Goze A. Devoret R (1977) Complementation pattern of iexB and recA mutations in E coli K12: mapping of tif-l, h, rB and iz,cA mutations. Moi Gen GeJ:et 157, 69--82 ~'arvanton GT, Seegwick SG (1982) Cloned truncated recA genes in Escaerichia coll. II. Effects of truncated gene products on in vivo recA+ protein activity. ~1ol Gen Genet 185, 99- ! 04 Larminat F, Defais M (1989) Modulation of the SOS response by truncated RecA proteins. Mol Gen Genet 216, 106-11"~ Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. In: Molecular Cloning: A Iaboratorv Manual. Cold Spring Harbor Laboratory, Cold Spring H~bor. NY Messing J (1983) New M13 vectors for cloning. In: ,~lethods in Enzymology Recombinant DNA. Academic Press, NY, vol 101 Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13. 8764-8785
29
Yanish-Perron C, Vieira J, Messing J (1985) Improved M I3 phage cloning vectors and host strains: nucleotide sequences of the M13mpl 8 and pUCI9 vectors. Gene 33, 103-119 30 Debarbouill6 M, Kuntz F, Klier A, Rapoport G (1987) Cloning of the SacS gene and coding a positive regulator of the sucrose regulon in Bacillus subtilis. FEMS Microbio141,137-140 31 Miller JH (1972) Experiments in molecular genetics. In: E.weriments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 32 Csonka LN, Clark AJ (1979) Deletions generatcd by the transposon Tn I 0 in the srl recA region of Escherichia coli K- 12 chromosome. Genetics 93, 321-343 33 Thomas R (1966) Control of development in temperate bacteriophage. I. Induction of prophage gene following heteroimmune superinfection. J Mol Bio122, 79-95 34 Blanco M, Levine A, Devoret R (i975) LexB: a new gene governing radiation sensitivity and lysogenic induction in Escherichia coli K-12. In: Molecular Mechanisms f o r Repair DNA (Hanawalt P, Stelow RB, eds) Plenum Press, NY, 379-382 35 Salles B, Weintstock GM (1989) Mutation of the promoter of LexA binding sites of tea, the gene encoding colicin E 1. Mol Gen Genet 215,483-489 36 Kobayashi N, Knight K, McEntee K (1987) Evidence for nucleotide mediated changes in the domain structure of the RecA protein in Escherichia coil Biochemiso'v 26, 6801-6810
,'oi
Site-directed m u t a g e n e s i s ir,l the Escherichia coil recA gene C Cazaux, F Larminat, M Defais* Laboratoire de Pharmacologie et de To.ricologie Fondamentales. CNRS, 205 route de Narbonne. 31077 Toulouse Cedex. France
(Received 19 October 1990: accepted 10 December 1990)
Summary - - Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its i~volvement in an ATP binding site, as previously shown in vitro [ 1|. RecA / recombination / SOS repair
Introduction
Materials and M e t h o d s
The first discovered role o f E coli R e c A protein was its r e c o m b i n a s e activity [2, 3]. However, R e c A has another p r i m a r y function in the control of the SOS s y s t e m [4-6] in which it acts as a protease cofactor 17! a l l o w i n g the cleavage o f L e x A repressor [81. Moreover, R e c A participates in the expression o f m a n y SOS functions such as post-replicative r e c o m b i n a t i o n [9, 10], repair o f double-strand breaks [1 1] or induced m u t a g e n e s i s [ 1 2 - 1 7 ] . To p e r f o r m these f u n c t i o n s , R e c A has to be activated b y interaction with singlestrand D N A and A T P [18]. These multiple functions o f R e c A protein suggest the presence o f several active sites in the protein w h i c h have been located on the r e c A gene according to the different mutant phenotypes [ 1 9 - 2 2 ] . P r e v i o u s genetic studies led to the c o n c l u s i o n that R e c A possessed different d o m a i n s for r e c o m b i n a t i o n a n d c o p r o t e a s e a c t i v i t y [23, 24]. R e c e n t l y it has been s h o w n that these d o m a i n s m a y overlap [22; O g a w a et al, u n p u b l i s h e d results]. In this study we attempted to define in the R e c A protein a d o m a i n o f interaction with either the L e x A repressor or with ATP, using specifically constructed mutations o f the r e c A gene, whose p h e n o t y p e s we s u b s e q u e n t l y e x a m i n e d in vivo.
Media and growth conditions
*Correspondence and reprints
Cultures were grown in Luria broth (Difco) at 37°C. Plasmidharbouring cells were always grown in the presence of I U U ~..L~IIIII dlll[.311,.,llltll ~,O1~111~1.]. IYIIU3 ~ l i n | m a | medium su emented with 0.4% glucose. 0.5% casamino acid and 1 lag/ml thiamine [311 was used for the 13-galactosidase assay. McConkey medium (Difco) supplemented with 1% lactose was used for the recombination assay. Oligonucleotide-directed mutagenesis ofrecA gene
A 3.l-kb fragment carrying the E coli recA gene and excised by BamHl digestion from the plasmid pFL352 [25] was cloned into the BamHI site of M 13mp 10 [26]. M 13mp 10/recA phages were selected and single-stranded phage DNA were purified as described by Messing 127].The recA mutants were prepared as described by Eckstein [28]. Mutagenic primers used to produce mutations were synthetized using DNA synthetiser 38!A (Applied Biosystems). Each mutation created a restriction site for facilitating the screening of the mutated phages. The duplex DNA was used to transform competent JM101 [29] or TG! cells [30]. Individual plaques were selected and phage DNA carrying the desired mutation were identified by digestion with the appropriate restriction enzyme. SOS induction
Colicin E1 induction with cea::lacZ fusions was nleasured as previously described [31], using the strains FL8641 [25] and FL8642, recA430.
282
C Cazaux et al
Dctermimttion q~flac recombination
Table I. Amino acid changes and mutant denomination.
intrachromosomal recombination was measured in FL7023 ~GY7023 ~'ecA) bacteria harbouring a recombinant plasmid or canwino the recA4 ¢0 mutation as described [ 171.
Mutated protein
Change in nucleotide
Change in protein sequence
RecA694 RecA430 RecA604 RecA605 RecA659 RecA611 RecA664
A T T - - > GTT GGT m > AGT GGT ~ > GCG G G T - - > GTT GAA - - > CAA GGT m > GCG TAC - - > GAG
lie (199) - - > Val Gly (204) - - > Ser Gly (204) - - > Ala Gly (204) - - > Val Glu (207) - - > Gin Glu (211) ~ > Ala Tyr (264) - - > Glu
Bacterhd amt phage assays The strain used was JC!0289 I321. Phages were lambda+ 1331. UV irradiation, cell survival and lambda phage survival assays ~ere carried out as described previously 141.
Results and Discussion In order to dissociate the different active sites of RecA protein we constructed a series of point mutations in the recA gene (table I). We first changed codon 839 coding for Tyr 264. This amino acid has been shown in rin-o to interact with ATP [ 1]. The replacing amino acid was Glu, bearing a negative charge compared to tyrosine and leading to the mutation recA664. Two mutations, recA604 and recA605, were created at the site of the recA430 mutation which is known to affect the coprotease activity of the protein, ie the l i k e l y site of i n t e r a c t i o n with L e x A r e p r e s s o r . recA430 mutants in which wild-type Gly 204 is replaced by Ser show normal recombinase activity I341. RecA604 and RecA605 proteins have comparable amino acid substitution, carrying 1 or 2 additional methyl ~ o u p s which yield the amino acids Ala and Val respectively. To further a.n.a!y~e the extent of R e c A - L e x A interaction we constructed 3 mutants, one of which, recA694, is located 5 amino acids upstream of the recA430 mutation. The last 2 mutations were respectively 3 and 7 amino acids downstream from the same mutation. In these cases substitution changed neither the charge, nor the hydrophobicity, nor the steric hindrance of the amino acids, which permitted us to analyse the effect of presumably minor modifications of the protein. These mutations were cloned in pBR322 and used to transform a ArecA strain. We then analysed the capacity of these new recA mutants to induce and express the SOS functions and to ca;ry out intrachromosomal recombination. Table II summarizes the results obtained with each mutant. The recA664 mutant behaved as a recA deletion strain. Tyr 264 had been previously shown to be essential to the in vitro binding of ATP to RecA protein [1]. Hence it is conceivable that changing Tyr into GIo destroyed the ATP binding site, leading in vivo to a complete loss of ar;y RecA activity. This finding is also in agreement wi:h the crystal structure of RecA, which indicates that ADP binds to Tyr264 and to amino acids 71-73 [Story and Steitz, unpublished results].
recA604 and recA605 mutations were located at amino acid 204. In these mutants we expected a phenotype analogous to that observed for the recA430 mutant, ie slight UV sensitivity, decreased SOS induction and normal recombination capacities. It can be seen in table II that adding 1 or 2 methyl groups to amino acid 204 affects UV sensitivity only slightly. The ability of these mutants to induce the SOS response was measured using a cea::lacZ fusion containing a ' p r o m o t e r up' mutation in order to increase sensitivity [25, 35]. The kinetics of induction of B-galactosidase activity were identical in all 3 mutants recA430, recA604 and recA605. Thus it appeared that the capacity of these mutants to promote LexA self-cleavage was decreased compared to the wild-type. These mutants were also deficient in SOS repair since they were unable to reactivate UV irradiated lambda phage and no mutagenesis could be detected in phage progeny (data not shown). This . . . . '-1 be ~. h. l.,.J l'-:---~ d l l l g ~ L I by ""-LIIC '~ lgltL, lat t:uuiu . . . t. t l'-~" the mutants are unable to derepress the SOS response, and therefore the functions necessary for Weigle repair were not transcribed. Alternatively, the mutant proteins may be unable to promote UmuD cle~'vage which is required for mutagenesis [ 16]. Intrachromosomic recombination was unaffected by the presence of one methyl group on residue 204. Table IL SOS and recombination activities. Strains
UV cea::lacZ Weigle resistance induction reactivation of lambda
Recombinant activity
RecA÷ RecA430
+++ +
RecA694 ++ RecA659 . RecA611 . RecA604 RecA605
+++ _
+++ ++
++
++ . .
+++
_ _
++ _
. .
+ +
RecA664 .
+++ + . . + + .
.
.
Site-directed re~'A mutants The r e c o m b i n a t i o n e f f i c i e n c y o f r e c A 6 0 4 mutant was i d e n t i c a l to that o b s e r v e d w i t h r e c A 4 3 0 m u t a n t . H o w e v e r , the addition o f a second m e t h y l group at this position led to a strong decrease in r e c o m b i n a s e activity without reaching a recA nul phenotype. T h e s e data indicate that a m i n o acid at position 204 w h i c h at first appeared to be i n v o l v e d only in the interaction with L e x A repressor could also be required for r e c o m bination. To further study the e x t e n s i o n of' L e x A interaction around residue 204, the mutation r e c A 6 9 4 , w h i c h is located 5 a m i n o acids u p s t r e a m from residue 204 and r e c A 6 5 9 and recA611 mutations, 3 and 7 a m i n o acids d o w n s t r e a m , were analysed. R e c A 6 9 4 protein s e e m e d to be only slightly affected in the SOS response and in recombination. However, recA659 and recArli mutants lost all R e c A functions and b e h a v e d as recA deletion strains, despite the fact that they produced n o r m a l quantities of the mutated protein as detected by S D S - P A G E (data not shown).
Conclusion It thus appears that residue 204 could be at the edge o f a site w h i c h extends at least 7 a m i n o acids d o w n stream. This residue plays a key role, since a slight modification o f the a m i n o acid structure has a d r a m a t i c e f f e c t on R e c A a c t i v i t y ( r e c A r 0 5 ) . T h e u p s t r e a m region m a y not be involved in any R e c A function. The d o w n s t r e a m J egion m a y interact not o n l y with L e x A r e p r e s s o r but p r o b a b l y also with ATP ~in¢'~ m,,tnnt S c h a n g i n g "~ Aiff. . . . , ~.~,~ . . . . ~n this region lose all R e c A functions - not only repair, but also recombination. O v e r l a p p i n g of A T P b i n d i n g and L e x A interaction d o m a i n s at a m i n o acids 200 and 205 has b e e n also d e m o n s t r a t e d by O g a w a et al ( u n p u b l i s h e d results). It cannot be confirmed by the c r y s t a l l o g r a p h i c structure d o n e in the p r e s e n c e o f A D P and in the absence o f D N A (Story and Steitz, u n p u b l i s h e d results) w h i c h could create a different c o n f o r m a t i o n a l c h a n g e t h a n that p r o v o k e d in the p r e s e n c e o f A T P and D N A [36]. . . . .
,
. . . .
~ v
l l i ~ l ~ i
~
'll,l ~ l I 1 1 ~ , ~ 1 ~¢~,'I i I~
I ~1.¢ II~l 1 ' l t ~ l I'~1 ~lm~ ~ I
I11
Acknowledgments The authors acknowledge the collaboration of H Mazarguil for synthesis of oligonucleotides. This work was supported in part by the Regional Council of Midi-Pyrrnres.
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