- Email: [email protected]
λ Repressor mutants that are better substrates for RecA-mediated cleavage
J. Mol. BioE.(1989) 206, 29-39
h Repressor Mutants that are Better Substrates for RecA-mediated Cleavage Frederick S. Gimblet and Robert T. Sauer Department of Biology Massachusetts Institute of Technology Cambridge, MA 02139, U.S.A. (Received 21 January 1988, and in revised form 8 August 1988) RecA-mediated cleavage of the bacteriophage 1 repressor results in inactivation of the protein and leads to induction of the 1 prophage. Here, we report the identifieation of three mutations in 1 repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the in& mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.
1. Introduction An important property of many transcriptional repressors is their ability to be inactivated in response to specific environmental signals. For example, when bacteriophage 1 lysogens are treated with DNA damaging agents, d repressor is cleaved in a RecA-dependent reaction and the prophage is induced (Roberts t Roberts, 1975; Roberts & Devoret, 1983). This cleavage reaction can be reproduced in vitro by incubating purified 1 repressor with a complex of RecA protein, singlestranded DNA, and ATP or an ATP analogue (Roberts et al., 1978; Craig & Roberts, 1980). The single-stranded DNA in the reaction is thought to mimic the inducing signal produced irk vivo by DNA damaging treatments. Although 1 repressor cleavage under physiological conditions is strictly dependent upon RecA protein, repressor cleavage at alkaline pH has been shown to be autocatalytic (Little, 1984). For these reasons, it is thought that RecA protein acts by stimulating the inherent autodigestion activity of I repressor. 3, repressor mutations that prevent prophage induction (ind- mutations) can be divided into two
groups based upon the properties of the corresponding mutant repressors (Gimble t Sauer, 1986). The first group includes mutant repressor proteins that are resistant to RecA-mediated cleavage and resistant to autodigestion at alkaline pH. Some of these group I mutations alter the residues bordering the site at which RecAdependent cleavage and autodigestion occur (Sauer et al., 1982; Little, 1984). The second group of indrepressors are also resistant to RecA-mediated cleavage but these repressors are able to autodigest at alkaline pH. We have argued that these group II mutations identify side-chains that are involved in binding of I repressor to the RecA protein (Gimble & Sauer, 1986). Here, we describe mutations that act as intragenic, second-site suppressors of a group II ind- mutation. Phages containing the ind- mutations and one of the second-site mutations are inducible in vivo and the corresponding revertant repressors are sensitive to RecA-mediated cleavage in vitro. Purified repressors bearing only the secondsite changes have properties similar to the previously described i&‘-l repressor (Cohen et al., 1981); i.e. these repressors are hypersensitive to RecAmediated cleavage and display a reduced ability to form dimers. We show that this reduced dimerization
t Present address: Department of Biochemistry, University of California, Berkeley, Berkeley, CA 94720, U.S.A. 0022-2$3ti/s9/050029-11
$03.00/O
is the
mechanism
changes improve
by
which
the efficiency
the
second-site
of RecA-mediated
cleavage.
29
0 1989 Academic Press Limited
30
F. A’. C&n&
2. Materials and Methods (a) Buffers Standard buffer (SB200) contains 10 miv-Tris HCl (pH 8), 0.1 mM-EDTA, 200 miw-ECl, 1.4 miw-2-mercaptoethanol, 5% (v/v) glycerol (SB50 has 50 m&r-ECl, SB800 has 800 mM-ECl). Lysis buffer is 100 mM-Tris. HCl 200 mM-ECl, 1 mM-EDTA. 2 mM-CaCl,, (PH 8) 1.4 m&r-2-mercaptoethanol. 5% (v/v) 10 mM-MgCl,, glycerol. RecA cleavage buffer contains 50 m&r-ECl, 15 mw-Tris. HCl (pH 7.5), 2 m&r-MgCl,, 2 m&r-dithiothreitol, 0.1 mM-EDTA. Assay buffer is RecA cleavage buffer supplemented with 100 pg bovine serum albumin/ml. (b) Isolation
of revertant
bacteriophages
Revertants of the in& bacteriophage 2 c1 GR185 (Gimble & Sauer, 1985) were isolated following ultraviolet light induction of a mutagenized population of lysogens. Hydroxylamine mutagenesis of Escherichia coli strain GW5100 (1 c1 GR185) was performed according to the procedure described by Miller (1972). Mutagenized cells were grown to logarithmic phase in LB broth, washed once, and resuspended in 5 ml of 0.01 M-MgSG,. Prophage induction was initiated by irradiating a 20-fold dilution (in 091 M-MgSG,) of this culture with (30 J/m’) ultraviolet light. A portion of the cells (1 ml) was diluted X-fold into LB broth, incubated at 37°C for 2 h, and killed by chloroform lysis. Part of this culture (0.1 ml) was plated with 0.2 ml of strain US3 (thi-, his-, la&-, ladYi, supa, recA - ) on Trypticase (BBL Microbiology Systems) plates and phage were allowed to grow overnight at 37°C. Turbid plaques were picked and spotted onto lawns of strain US3 and strain DM1187 (F-. his-4, recA441, s&All, lezA3-51; Mount, 1976). The increased level of activated RecA protein in strain DM1187 results in a clear plaque phenotype for ind+ pseudorevertants or wild-type phage and a turbid plaque phenotype for ind- phage (Mount, 1976). Candidates from 3 independent, mutagenesis experiments, which yielded clear plaques on strain DM1187 and turbid plaques on strain US3, were isolated and plaque-purified twice on strain DM1187. (c) Construction revertant
of plasmids subditutions
bearing
Restriction digestions, ligations and gel electrophoresis were performed by standard procedures (Maniatis et al., 1982). The restriction fragments used for cloning were purified by electrophoresis on low melting point agarose gels (NuSieve, FMC Corp.) or were obtained by electroelution from polyacrylamide gels. The c1 (repressor) gene from the revertant 1 phages was initially cloned into plasmid vector pZ152 (Zagursky & Berman, 1984). Purified 1 DNA from each of the revertants was digested with EcoRI and PvuI and ligated to a preparation of EcoRI and PvuI-cut pZ152. After transformation into E. coli strain X90 (P’2acIq’ pro, ara-, A(Zac-pro)l3, n&A, argEam, thi-, rif), colonies resistant to tetracycline (10 pg/ml) and to infection by ICI- phage were selected. The presence of the c1 gene in candidate plasmids was confirmed by restriction analysis. A 1.1 kbt fragment bearing the c1 gene was purified from a BglII and Pet1 digest of these plasmids and was cloned into t Abbreviations polyethyleneimine.
used: kb, lo3 base-pairs; PEI,
and R. T. Sauer M13mp8 for “dideoxy” sequence analysis @anger pf a/.. 1977). DNA sequencing revealed that each of the reverted cl genes contained the original Gly185+Arg (GRlS5) indmutation, a Leu64-+Met (LMM) mutation, and one of 3 revertant substitutions (Met87-+Thr. MT87: Alal52+Thr, AT152; or Prol58-+Thr. PT158). Wc assumed that the LM64 mutation was silent,: and indeed. found that it had been overlooked in the original sequencing of the GR185 ind- mutant. Subcloning and/or cassette mutagenesis were used to remove the LM64 mutation and to construct overproducing plasmids containing c1 genes with: (1) the GR185 ind- mutation alone; (2) the GR185/MT87, GR185/AT152, or GR185/PT158 double mutations; and (3) the MT87. AT152 or PT158 mutations alone. To confirm thr presence of the proper mutations in these plasmids. the entire CT gene was sequenced. The structure of each plasmid is identical and contains a 164 kb EcoRI -0laI fragment carrying a P,,,-i repressor fusion (Backman & Ptashne, 1978) cloned between the EcoRI and C:ZaI sites of plasmid pZ150 (Zagursky & Berman, 1984). Plasmids pFG610 and pFG611 are P,,, derivatives of the MT87 and PT158 plasmids described above. (d) Inactivation
of mutanf
repressors
in vivo
The sensitivity to RecA-mediated inactivation of mutant repressors borne on plasmids was assayed by phage spot tests in the presence or absence of 0.7 p’g mitomycin C/ml (Gimble & Sauer, 1985). (e) Protein purijcations The wild-type and mutant repressor proteins were purified essentially as described by Johnson et al. (1980). Repressor proteins labeled with [35S]methionine were purified using a modification of this method. Strain X90 containing plasmids pMH236 (encoding wild-type repressor; Hecht et al., 1986) pFG610, or pFG611 was used for the purifications. Cells were grown in M9 minimal medium (100 ml) containing ampicillin (100 pg/ml), arginine (40 pg/ml) and vitamin Bl (1 pg/ml) (Miller, 1972) at 37°C to an approximate concentration of 5 x 10s cells/ml. The culture was induced by the addition of 100 pg isopropyl-1-thio-/I-n-galactoside/ml, grown for an additional 20 min and then labeled with [35S]methionine (5 mCi, 1000 Ci/mmol; New England Nuclear) for 5 min. After the cells were harvested by centrifugation, they were resuspended in lysis buffer to a concentration of 10 mg/ml, lysed by sonication, and centrifuged at 4°C for 1 h at 14,000g. The supernatant was decanted and nucleic acids and cell debris were precipitated by adding polyethyleneimine (PEI) to 0.20/, (v/v) followed, by centrifugation. Two volumes of a saturated solution of (NH&SO, were added to the PEI supernatant, the solution was stirred overnight, and the protein pellet was collected by centrifugation at 16,000g for 1 h. The pellet was resuspended in 16 ml SB50 buffer/g of cells, and the solution was dialyzed extensively against SB50. The dialysate was loaded onto a 1 cm x 8 cm column of CMSephadex (C-50; Pharmacia Fine Chemicals) for wild-type and PT158 repressors, or onto a 1 cm x 8 cm column of Affi-gel blue (Bio-Rad) for the MT87 protein. The columns were washed with SB50 and repressor was eluted with SB300 for the CM-Sephadex columns and SB800 for the Affi-gel blue column. Fractions containing labeled repressor were pooled and loaded onto a 1 cm x 8 cm column of hydroxylapatite (Clarkson Chemical Co.) that
1 Repressor
had been equilibrated in 0.2 M-potassium phosphate (pH 8.0). The columns were washed with the same buffer and repressor wm eluted using a single step of 1.0 M-potassium phosphate (pH 8-O). The fractions containing repressor were pooled, concentrated by ultrafiltration (YM-10 membrane; Amicon Corporation), and then dialyzed against RecA cleavage buffer. The specific radioactivities of these repressors were estimated to be 2 x 1016 cts/min per mol by scintillation counting and SDS/polyacrylamide gel electrophoresis. The disulfide-bonded YC88 dimeric repressor and the YC88 repressor, which had been reduced and alkylated with iodoacetamide, were gifts from John ReidhaarOlson. RecA protein and the IS84 repressor were gifts from Dr Kendall Knight and Kathy Hehir, respectively. The C-terminal 93-236 repressor fragment was purified from strain X90 bearing plasmid pFG401. This plasmid encodes i repressor residues 93 to 236 under transcriptional control of a tat promoter (Gimble, 1987). The 93-236 fragment was purified by ion exchange chromatography on QAE-Sephadex and DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose, and gel chromatography using Ultrogel AcA44. filtration Sequential Edman degradation confirmed that the purified fragment had the N-terminal sequence Met-GlnPro-Ser-Leu expected for a fragment starting at residue 93 of int,art repressor (Gimble, 1987). ( f) RecA-mediated cleavage and autodigrstion RecA-mediated cleavage of wild-type or mutant repressors was generally assayed by incubating samples containing 4.5 PM-repressor, 2.7 q-Red protein, 1 miv-adenosine 5’-[y-thio]triphosphate (BoehringerMannheim), 4.7 pg poly(dT)/ml (PL Biochemicals), in RecA cleavage buffer at 37°C. Portions of the samples (30 ~1) were withdrawn at several time points and the reactions were stopped by the addition of 10 ~1 of SDS sample buffer and heating to 90°C. When RecA-mediated cleavage of the YC88 dimer was analyzed, reducing agent was omitted from the reaction mixtures and from the SDS sample buffer, and 1 ~1 of a 1 M solution of iodoacetamide was added to each reaction immediately prior to addition of SDS sample buffer to block sulfhydryl-disulfide rearrangements. Autodigestion reactions were performed using a modification of the methods described by Little (1984). Samples containing 3.2 PM-repressor, 50 mM-KCl, and 50 mM-CAPS. NaOH (pH 10.5) were incubated at 37°C. and portions (30~1) were removed at various times. The reactions were stopped by the addition of 10 ~1 of SDS sample buffer and by heating. The extent of cleavage in most autodigest,ion and RecA-mediated cleavage experiments was determined by running samples on SDS/ urea/l 80,;) polyacrylamide gels (Ito et al.. 1980) and staining with Coomassie brilliant blue. Stained gels were scanned using au LKB 2202 ultroscan laser densitometer equipped with an LKB 2220 recording integrator. To quantify the extent of cleavage, the peak areas of the 2 digestion fragments were added and calculated as a percentage of the total repressor area, which is the sum of the peak areas of the fragments and of the remaining intact repressor. Rates of RecA-mediated cleavage of wild-type or mutant repressors at different total repressor concentrations were determined using a modification of the procedure of Cohen et aZ. (1981). Samples containing 5 nm-[“5S]repressor, 1 rnM-adenosine 5’.[y-thioltriphosphate. 100 pg bovine serum albumin/ml in RecA
Cleavage
31
cleavage buffer were incubated at 4°C for 30 min in the presence or absence of 1 PM-unlabeled repressor of the same type. After the solution was heated for 5 min at 37°C. it was combined with a solution of 54pm-RecA protein and 9.3 pg poly(dT)/ml, which had been preincubated for 30 min. Portions (30 ~1) of each reaction mixture were withdrawn at different times, the reactions were stopped by heating in SDS sample buffer, and the samples were electrophoresed on 15y0 protein gels (Dreyfus et aZ., 1984). To visualize the repressor and fragment, bands, small amounts of unlabeled repressor or cleavage fragments were added to each mixture prior to electrophoresis. After the gels were stained and destained, the bands containing intact repressor and the 2 fragments were excised, placed into different scintillation vials containing 9 ml of Soluscint-0 (National Diagnostics), 1 ml of Hydrofluor (National Diagnostics) and 0.5 ml Solusol (National Diagnostics), heated at 50°C for 2.5 h, and then left, overnight before counting. The degree of cleavage was calculated as the ratio of the radioactivity in the 2 fragment bands to the radioactivit,? in the 2 fragment bands and the repressor band. (g) Nitrocellulose Jilter binding of operator DNA experiments Nitrocellulose filter-binding were performed in assay buffer using the general methods described by Gimble & Sauer (1986). A restriction fragment containing the lORl site was used as the source of operator DNA (Gimble & Sauer, 1986). Experiments were done in triplicate, and the repressor concentrations at half-maximal binding were calculated by computer curve-fitting of the data using linear regression. (h) Gel filtration
chromatography
The apparent molecular weights of repressor oligomers were determined by gel filtration chromatography. Samples (200 pl) containing repressor at a concentration of 4 pM and a myoglobin standard were injected onto a 0.75 cm x 60 cm Bio-Sil TSK-125 HPLC gel filtration column (Bio-Rad Laboratories) and were chromatographed in a buffer containing 15 mill-Tris. HCI (pH 7.5), 50 mM-KCl, 0.1 mM-EDTA, 2 mM-M$l,. All experiments were performed at 37 “C. Protein peaks were detected by ultraviolet absorbance at 280 nm. The columns were calibrated with bovine serum albumin, ovalbumin, carbonic anhydrase. myoglobin and vitamin B12.
3. Results (a) Isolation and sequence analysis of revertants A I prophage bearing an ind- mutation cannot be induced because its mutant repressor cannot be inactivated by RecA-mediated cleavage (Roberts t Roberts, 1975). As a consequence, revertants can be isolated by inducing a mutagenized culture of an indlysogen with ultraviolet irradiation and screening among the small number of progeny phage for those having an ind+ phenotype. We used a selection and screening method of this type to isolate three independent revertants of the indphage Iz CT GR185 (see Materials and Methods). The revertant repressor genes were cloned into plasmids, and mutations were located by DNA sequencing.
3%
F. S. Nimble and R. T. Sau,er
Table 1
Table 2
Second site mutations that restore an ind+ phenotype to th,e GR185 repressor Code
Second-site mutation
MT87
Met87 +Thr ATG ACG Ala152 +Thr GCA ACA Pro158 +Thr ACA I, CCA
AT152 PT158
of mutant repressors in vivo
Indu,cibility
Each revertant retained the original ind- mutation (GR185) and a silent mutation (LM64) that was present in the parental ind- phage. In addition, each revertant acquired a second-site mutation (MT87, AT152, or PT158; see Table 1). To avoid complications in characterizing the revertants, we constructed overproducing plasmids containing repressor genes without the silent mutation. We also constructed repressor genes in which the second-site mutations were present in otherwise wild-type backgrounds. (b) RecA-mediated cleavage properties of the mutants in vivo The sensitivity to RecA-mediated cleavage in ~ivo of the revertant repressors and repressors bearing only the second-site mutations was assayed by phage spot tests in the presence and absence of the inducing agent mitomycin C (see Table 2). In the absence of induction, each of the plasmid-borne repressors listed in Table 2 prevents lytic growth of the infecting phage. This indicates that each repressor is able to bind to operator DNA. The strain containing the GR185 ind- repressor is as resistant to phage infection under inducing conditions as under non-inducing conditions. As noted
Spot phenotype ( -MC) Induced ( + MC)
Uninduced
Allele
(‘Iear Turbid Turbid Turbid
No reprevsor Wild-type GR185 GR185/MT87 GR185/AT152 GR185/PT158 MT87 AT152 PTl58
‘hbid
(:lear Semi-turbid Turbid Semi-turbid Semi-turbid &m-turbid
Turbid Turbid Turbid
( kar (Year
Turbid
(:ll%L:
For details of the assay, see Gimble & Sauer (1985). +MC, 0.7 pg mitomycin C/ml.
earlier, this results from the resistance of the indrepressor to RecA-mediated cleavage (Gimble & Sauer, 1985). Strains containing wild-type repressor and the revertant repressors (GR185/AT152, GR185/PT158, GR185/MT87) are somewhat sensitive to phage infection under inducing conditions. This suggests that these repressors can undergo RecA-dependent cleavage in vivo. Finally, in the presence of mitomycin C, strains containing repressors that bear only the second-site mutations (MT87, AT152, PT158) behave like strains that contain no repressor; i.e. they are extremely sensitive to phage infection. This result suggests that repressors bearing only the second-site changes are more sensitive to RecA-mediated cleavage in vivo than wild-type repressor. (c) RecA-mediated mutant
cleavage of puri$ed. repressors
Wild-type repressor, the GR185 ind- repressor, revertant repressors bearing the GRI 85/MT87,
Wild type
GRl85
20
i-----J-t 20 240 0
PTl58
GRl85lPTl58
---II
L 0
240
J 0
20
240
0
20
240
Time (min)
Figure 1. Analysis of RecA-mediated cleavage of mutant and wild-type repressor proteins by SDS/polyacrylamide gel electrophoresis. The extent of RecA-mediated cleavage for the wild-type, GR185, GRlf%/P’I’158, and PT158 repressors is shown after 0, 20, and 240 min of incubation at 37°C. Intact repressor is indicated as IR, and the products of digestion are indicated as irl (residues 112 to 236) and dr2 (residues 1 to 111).
A Repressor Cleavage
repressor monomers (R) relative to dimers (R,) by changing the dimerization constant K, . According to the model, only the repressor monomer binds to and is cleaved by RecA (Phizicky & Roberts, 1980) and an increase in the monomer concentration would increase the observed rate of RecA-mediated cleavage as long as RecA was not saturated. As we discuss below, the suppressor mutations decrease the ability of repressor to form dimers. Mutations could also increase the rate of RecA-mediated cleavage by increasing the rate of turnover of the RecA-R complex (increasing /&) or by increasing the affinity of the repressor monomer for RecA (decreasing the dissociation constant, KS).
GRl&/AT152 and GRl&/PT158 changes, and repressors bearing the single MT87, AT152 and PT158 changes were purified and assayed for sensitivity to RecA-mediated cleavage in vitro. As shown in Figure 1, RecA-mediated cleavage of the GR185 repressor is extremely slow and only trace amounts of cleavage products are seen after four hours of digestion. By contrast, wild-type repressor, the revertant repressors, and repressors containing only the second-site mutations are cleaved at much faster rates. Figure 2 shows time-courses of RecAmediated digestion for each of the repressor proteins. The GRl85/AT152 and GRl85/MT87 mutants are digested somewhat more slowly than wild-type, whereas the GR185/PT158 repressor is digested slightly faster than wild-type. Strikingly, the MT87, AT152 and PT158 repressors are digested at rates sixfold to Sl-fold faster than the wild-type protein under these conditions (Table 3). The relative order of cleavage rates is the same (PT158>AT152>MT87) whether the ind- mutation is present or absent, although cleavage of each protein is about 20-fold to 30-fold faster in the absence of the in& substitution. There are three simple ways in which the suppressor mutations might increase the rate of RecA-mediated cleavage of repressor. These can be discussed in terms of the following model for RecAmediated cleavage: KS k RecA + R o RecA: R * RecA + fragments
A
li Kl 0 R2 mutation
could
increase
the
(d) Autodigestion of the mutant repressors The repressor autodigestion reaction at high pH and the RecA-mediated cleavage reaction are thought to share a common catalytic mechanism (Little, 1984, Slilaty et al., 1986; Gimble & Sauer, 1986). Thus, the rate of autodigestion should be related to the rate of turnover of the RecA-R complex. To test for possible effects of the suppressor mutations on the catalytic rate constant (lcJ, we examined the rates of autodigestion for the mutant repressors. Table 3 shows that the revertants and the repressors bearing the suppressor mutations autodigest at rates that are very similar to the wild-type rate, Hence, the revertant substitutions do not appear to increase the rate of RecA-mediated cleavage by increasing the catalytic rate constant. It should be noted, however, that this conclusion could be incorrect if the detailed mechanisms of cleavage are different for autodigestion and RecA-mediated cleavage.
(1)
population
33
of
too
- 7
/
/
MT07
PTl58lGRl85.
y
80
120
ATl52/GR185,
180
240
Time (mid
Figure 2. Kinetics of RecA-mediated cleavage of mutant and wild-type repressors. The purified proteins were digested for different times at 37”C, and the extent of cleavage at each time point was measured by SDS/polyacrylamide gel electrophoresis as described in Materials and Methods.
F. S. Nimble and R. T. Sauer
34
Table 3 Ret A -mediated cleavage and autodigestion RecA-mediated cleavage? (relative rate)
Allele
Autodigestion$ (relative rate)
la
l@§
Wild-type GR185 GR185/MT87 GR185/AT152 GR185/PT158 MT87 AT152 PT158
< 0.05 0.2 0.4 1.6 6.0 9.2 31.1
1.1 0.9 0.9 0.8 0.9 0.8 0.9
15.0 8.0
n.d. n.d.
IS84 93236 fragment
n.d., not determined. t Reaction mixtures contained 4.5 par-repressor, 2.7 pa-RecA protein, 1 mM-adenosine 5’-[y-thioltriphosphate, and 4.7 pg poly(dT)/ml in RecA cleavage buffer. $ Reaction mixtures contained 3.2 fin-repressor, 50 mM-KCl, and 50 mM-CAPS NaOH (pH 10.5) and were incubated at 37°C. Q The initial rate of RecA-mediated digestion of wild-type repressor is 39 ng/min per pg RecA protein. 11The wild-type rate constant for autodigestion is 166 x 10-a 6-i.
(e) Dimerization of the mutant repressors To study repressor dimerization, gel filtration chromatography was used to compare the elution positions of wild-type repressor and the MT87, AT152 and PT158 repressors (Table 4). Because monomers and dimers of repressor are in rapid equilibrium in such experiments (Sauer, 1979), the proteins elute as trailing peaks and a rigorous analysis of the monomerdimer equilibrium is not if each possible (Ackers, 1970). Nevertheless, repressor is loaded at the same initial concentration and is chromatographed under the same conditions, then it should be possible to judge the relative ability of each of the repressors to dimerize. Under the chromatographic conditions of these experiments, wild-type repressor elutes at an apparent molecular weight of 41,000. This value is intermediate between those expected for a monomer (26,000) and a dimer (52,000). The MT87, AT152 and PT158 proteins elute at apparent molecular weights that are from 4000 to 8000 less than the apparent molecular weight at which wild-type repressor elutes (Table 4). Thus, each of these
mutations appears to impair the ability of repressor to dimerize. Because repressor dimers are the oligomeric species that bind to operator DNA (Chadwick et al., 1970; Sauer, 1979), a mutant displaying reduced dimerization should also display reduced operator binding activity. This is clearly the case for the AT152 and PT158 repressors, which have approximately l/7 of the wild-type binding activity as assayed by nitrocellulose filter binding (Table 4). If these mutations affect DNA binding only by reducing dimerization, then the observed reduction in DNA binding activity would indicate an approximate 50-fold increase in the equilibrium dissociation constants for dimerization of the AT152 and PT158 repressors. The MT87 repressor shows no operator binding activity in the assay used (Table 4), but this result is difficult to interpret as other mutant repressors bearing N-terminal substitutions have been shown to have operator binding activity which is not detected by the filter binding assay (Nelson & Sauer, 1986). In fact, the MT87 mutant displays approximately the same operator binding activity in Z&JOas the AT152 and
Table 4 Effects on dimerization Allele Wild-type MT87 AT152 PT158
and operator binding
Apparent molecular weight
Relative operator binding activity?
41,000 37,000 37,000 33,000
1.0 Not detected 0.14 0.14
t Defined ae the concentration of wild-type repressor required to give half-maximal operator binding divided by the concentration required to give half-maximal operator binding for the repressor tested. Assays were performed by the nitrocellulose filter aaaay as described in Materials and Methods.
1 Repressor
100
(a) G
”
--
,n-Q hi
,oow
, Wild +w ,
PT158
35
Cleavage
The model also predicts that the concentration dependence of the relative cleavage rate should be reduced for mutants that dimerize less well than wild-type. Figure 3 shows experiments of this type for high (1 PM) and low (5 nM) concentrations of wild-type repressor, the PT158 repressor and the MT87 repressor. These experiments were performed with total RecA protein in excess over total repressor to avoid complications caused by saturation of RecA protein. For wild-type repressor, the relative rate of cleavage is about fourfold faster at the low than at the high concentration. The relative rate of cleavage of the MT87 repressor is about twofold faster at the low concentration. and that of the PT158 repressor is the same at the low and high concentrations. These data are therefore consistent with the model and the idea that the MT87 and PT158 mutations reduce the ability of repressor to dimerize. The dimerization constant for wild-type repressor is approximately 20 nM (Sauer, 1979) and, in the absence of RecA protein, approximately lo?/, of the repressor should be monomeric at a concentration of 1 pM and 70% should be monomeric at a concentration
I 60
I 30 Time
I ‘90
(min)
of 5 nM. Thus, a sevenfold
difference in the relative initial rates at’ the low and high concentrations would be expected if the monomer-dimer equilibrium were not perturbed by RecA binding. The observed fourfold differences in rates would be expected if the RecA-repressor monomer affinity constant (K,) were approximately 3 PM. This value should be viewed as an extremely rough estimate, however, as the data are somewhat uncertain and the calculation depends upon several assumptions.
Figure 3. Kinetics of RecA-mediated cleavage of mutant and wild-type repressors at 2 different repressor concentrations. RecA-mediated cleavage of purified proteins was carried out at 37 “C as described in Materials and Methods using initial repressor concentrations of 5 mu (closed symbols) and 1 PM (open symbols). In these experiments, the concentration of RecA protein was repressor; (b) PT158 repressor: 5.4 PM. (a) Wild-type (c) MT87 repressor.
PT158 mutants, suggesting that it has binding activity in vitro which is not detected by the filter binding assay. Overall, the gel filtration and operator binding experiments are consistent and indicate that the MT87, AT152 and PT158 mutations interfere with repressor dimerization. (f) Effects of repressor concentration on RecA-mediated cleavage
The model of equation (1) predicts that the relative rate of RecA-mediated cleavage of wildtype repressor should be faster at low concentrations, where repressor is predominantly monomeric, than at high concentrations, where repressor is predominantly dimeric (Phizicky & Roberts, 1980).
(g) Additional
tests of the model
(i) Cleavage of other mutants reduced dimerization
displaying
If repressor dimers cannot be cleaved by RecA (model of eqn (l)), then most mutants displaying reduced dimerization should also display increased rates of RecA-mediated cleavage relative to wildtype. To test this prediction, we used two repressors bearing mutations that were not selected as revertants of an ind- phenotype. The first mutant contains the IS84 (Ile84+Ser) missense mutation. IS84 was originally isolated as a mutant defective in DNA binding and the protein was subsequently shown to be defective in dimerization (Hecht et al., 1983; Weiss et al., 1987). The second mutant contains repressor residues 93 to 236 but is deleted for the N-terminal domain of repressor. The 93-236 fragment dimerizes less efficiently than wild-type repressor because it is missing the N-terminal dimerization contacts (Pabo & Lewis, 1982; see Discussion). The data of Figure 4 and Table 3 show that the IS84 mutant and the C-terminal 93-236 fragment undergo RecA-mediated digestion considerably faster than the wild-type repressor and somewhat
F. 8. Gimble and R. T. Sau.rr
36
0
RecA
b
YC88 dwner
Wild type -r-----Ii.----c d
YC88 monomer
e
f
g
h
0
240
0
240
* ,:* 2’ I
Rearessor
Repressor fraqments r
--II 0
240
Time (min) 30 Time (min)
Figure 4. Kinetics of RecA-mediated cleavage of the 93-236 repressor fragment, the IS84 and MT87 repressors, and wild-type repressor. The purified proteins were digested at 37°C for different times as described in Materials and Methods.
faster than the MT87 repressor. Therefore, repressor mutants displaying reduced dimerization are more sensitive to RecA-mediated cleavage even in cases where the mutation was not selected on the basis of reverting an ind- phenotype.
Figure 5. Analysis of RecA-mediated cleavage of wildtype repressor, the disulfide-bonded YC88 dimer, and reduced and alkylated YC88 by SDS/polyacrylamide gel electrophoresis. The extent of RecA-mediated cleavage after incubation at 37°C for 0 or 240 min is shown for wild-type repressor (lanes c’ and d); disulfidr-bonded YC88 dimer (lanes e and f); and reduced and alkylated YC88 (lanes g and h). Lanes a and b show the wild-type repressor and the disulfide-bonded YC88 dimer, respectively, in the absence of RecA protein. Alkylation of Cys88 causes repressor and its digestion products to migrate more slowly than the analogous unmodified repressor and fragments (compare lanes d and h). A small amount of unalkylated C-terminal fragment is observed in lane h.
(ii) Cleavage of a covalent repressor dimer According to the model represented in equation (1 ), a repressor that is always dimeric shotild not undergo RecA-mediated cleavage. We have tested this prediction by using a repressor that contains a substitution (Tyr8GCys: YC88) that permits a disulfide bond to form between the two monomer subunits (Pabo & Suchanek, 1986; Sauer et al., 1986). Figure 5 shows that after incubation with RecA for four hours, half of the wild-type repressor has been cleaved but no detectable cleavage of the disulfide-bonded YC88 dimer has occurred (compare lanes d and f). Thus, the covalent dimer is extremely resistant to RecA-mediated cleavage. It is unlikely that this resistance is caused by any gross conformational change in the protein because the disulfide-bonded YC88 dimer binds strongly to operator DNA (J. Reidhaar-Olson, personal communication) and underdoes autodigestion at the same rate as wild-type repressor (data not shown). To control for the possibility that the YC88 mutation interferes directly with RecA-mediated cleavage, we reduced the disulfide bond and alkylated Cys88 with iodoacetamide prior to incubation with the RecA protein. This modified YC88 protein is sensitive to RecA-mediated cleav-
age (Fig. 5, lane h) and, in fact, is cleaved faster than wild-type repressor (compare lanes d and h). The faster cleavage of the alkylated YC88 protein probably results from reduced dimerization, as residue 88 forms part of the dimer interface of the N-terminal domain of repressor (Pabo & Lewis, 1982; see Discussion).
4. Discussion We isolated three second-site mutations that suppress the effects of a group II ind- mutation in 1 repressor. In the cell, the indrepressor is insensitive t,o RecA-mediated cleavage, revertant repressors containing the indmutation and a suppressor mutation are sensitive to cleavage, and repressors containing only the suppressor mutations are hypersensitive to cleavage. Similar phenotypes are observed in studies using purified RecA and repressor proteins. The indrepressor is not detectably cleaved in the RecA-mediated reaction in vitro, repressors bearing the ind- mutation and a suppressor mutation are cleaved at rates similar to wild-type, and repressors bearing only the
II Repressor
suppressor mutations are cleaved at rates significantly faster than wild-type. Thus, reversion analysis has allowed us to identify residue changes that make 1 repressor a better substrate for RecAmediated cleavage. Similar strategies of intragenic reversion have been used to identify sequence changes that enhance the affinity and specificity of 1 repressor-operator binding (Nelson & Sauer, 1985) and sequence changes that enhance the thermodynamic stability of staphylococcal nuclease (Shortle & Lin, 1985; Shortle, 1986). How do the suppressor mutations increase the rate of RecA-mediated cleavage of 1 repressor’! We believe that the main effect of the MT87, AT152 and PT158 mutations is to interfere with repressor dimerization. At concentrations where wild-type dimers normally predominate (above 20 nM; Sauer, 1979), each of the suppressor mutations causes an increase in the fraction of repressor present as monomer. By hypothesis, this should increase the rate of cleavage because the repressor monomer is the true substrate in the RecA-mediated reaction. Phizicky & Roberts (1980) originally proposed that the repressor monomer was the preferred substrate for RecA-mediated cleavage and our results provide strong support for their idea. First, each of the repressors containing a suppressor mutation alone displays reduced dimerization and undergoes RecAmediated cleavage faster than the wild-type repressor. Second, repressor mutants which display reduced dimerization but which were not selected on the basis of an Ind phenotype also exhibit enhanced rates of RecA-mediated cleavage. Third, a disulfide-linked repressor dimer is not cleaved in the RecA-mediated reaction even though it undergoes autodigest.ion at a rate indistinguishable from wildtype. Do the suppressor mutations affect RecAmediated cleavage solely by affecting repressor dimerization? We cannot answer this question rigorously because we have not determined equilibrium dimerization constants for the MT87, AT152 and PT158 mutants or kinetic parameters (K,,, and V,,,) for RecA-mediated cleavage of these repressors. However, at low concentrations where most repressor molecules should be monomeric, the MT87 and PT158 repressors are cleaved only slightly faster than wild-type repressor (Fig. 3). This indicates that these mutations act mainly by affecting repressor dimerization, and allows us to argue that the PT158 mutation, which is the strongest suppressor, interferes with dimerization to a greater extent than the MT87 mutation. This ranking is consistent with the gel filtration experiments. The MT87 and AT152 changes appear to have approximately the same effect on dimerization as assayed either by RecA-mediated cleavage at high repressor concentrations or gel filtration. The overall ranking from strongest to weakest dimerization would thus appear to be wildtype > MT87 = AT152 > PT158. The operator DNA binding experiments give a somewhat different order, but this may simply indicate that the
Cleavage
37
mutations affect operator binding directly as well as via dimerization. Because the suppressor mutations decrease dimerization, it is reasonable to ask if the original GRl85 itimutation increases repressor dimerization. Previously, we found that the GR185 mutation increased operator binding slightly, consistent with an approximate threefold increase in dimerization (Gimble & Sauer, 1986). We argued, however, that this increase was not sufficient to fully account for the resistance of the mutant to RecA-mediated cleavage and suggested that the GR185 change also affected RecA binding. How good is this argument? On the one hand, it is vulnerable because the change in DNA binding caused by the Gly185+Arg mutation may not accurately reflect changes in dimerization. On the other hand, we have also found that repressors with Glu185 and Va1185 are also ind- (Gimble & Sauer, 1985, 1986; J. Hu 6 R. Sauer, unpublished results). It is difficult to imagine how the side-chains of these three amino acids, which are chemically dissimilar, could all mediate improved repressor dimerization. By contrast, it is easy to envision how each sidechain could interfere with the binding of RecA. Hence, we feel that our original conclusion is probably correct, i.e. the GR185 mutation influences both dimerization and RecA binding. This issue could presumably be addressed directly by studying the RecA-mediated cleavage of the GR185 mutant at low repressor concentrations. The increased RecA-mediated cleavage activity reported here for the repressors bearing the suppressor mutations (MT87, AT152 and PT158) has also been observed in the case of the ind”-1 and iti”-2H mutant repressors (Crow1 et al., 1981; Cohen et al., 1981). Lysogens containing these 1 ind” alleles are hypersensitive to inducing treatments (Horiuchi & Inokuchi, 1987), and the id-1 repressor, like the mutants described here, dimerizes less well than the wild-type repressor (Cohen et al., 1981). The ,ind”-1 mutation is a Glu233+Lys substitution, close to the carboxyl terminus of repressor (Gimble & Sauer, 1985). How might the suppressor mutations affect dimerization? Intact 1 repressor contains two apparently distinct sets of dimerization contacts. The isolated N-terminal domain (residues 1 to 92) forms dimers in the crystal structure and in solution (Pabo & Lewis, 1982; Weiss et al., 1987). These N-terminal dimers are stabilized by hydrophobic helix 5-helix 5’ packing interactions which include the packing of Met87 against Ile84’, and Ile84 against Met87’. The MT87 suppressor mutation (Met87-+Thr), would be expected to reduce the free energy of N-terminal dimerization in two ways. First, the hydrophilic hydroxyl group of Thr87 would be introduced into the hydrophobic interface. Second, the S6 and GE groups of Met87, which contribute directly to the packing interface, would be removed. Thus, the crystal structure of the N-terminal domain allows us to rationalize the reduction in repressor dimerization that’ is caused
by the MetS’I-+Thr substitution. Similar arguments can be advanced to explain the reduced dimerization caused by the Ile84-+Ser mutation. The structure of the C-terminal domain of L repressor is not known, but a C-terminal fragment consisting of residues 132 to 236 forms dimers in solution (Pabo et al., 1979). Because the AT152 (Ala152-+Thr) and PT158 (Prol58+Thr) suppressor mutations are close in the primary sequence, it seems likely that this general region of the C-terminal domain plays an important role in mediating dimerization of the C-terminal domain. The wild-type Ala152 and/or Pro158 side-chains might form part of a hydrophobic dimer interface. In this case, substitution of either residue by threonine would be expected to disrupt dimerization in a manner analogous to that described for the MT87 mutation. Alternatively, the AT152 and/or PTI 58 mutations might cause conformational changes that affect dimerization indirectly. It seems clear that the repressor strongly preferred substrate for
monomer is the RecA-mediated
cleavage but the mechanism by which the dimer is resistant to cleavage is less clear. The simplest model is that RecA cannot bind dimers because the binding surface is occluded or altered by dimerization, but there are no direct experiments that show that RecA is unable to bind the repressor dimer. Alternatively, dimer-RecA complexes might form but, be unable to undergo cleavage. Slilaty et al. (1986) have suggested that repressor dimers do not autocleave, based upon their finding that the rate of autocleavage shows a concentration dependence under certain conditions. This would be consistent with the idea that dimer-RecA complexes might, not undergo cleavage. In fact, two of our mutations that affect dimerization, AT152 and PT158, are close in the primary sequence to Ser149, which is thought to play a catalytic role in the cleavage reactions (Slilaty & Little, 1987). As a result, it is plausible that some of the active site residues could be buried or occluded by repressor dimerization. On the other hand, we have observed that the disulfidebonded YC88 repressor dimer autocleaves at a rate similar to wild-type, which would seem to argue that the active site cannot be inaccessible in the dimer. A problem, here, is that we do not know that the C-terminal domains of the covalent dimer are actually in contact under the conditions (pH 10.5, 37°C) where our autocleavage experiments were performed. This issue needs to be studied further. Moreover, to directly address the issue of why repressor dimers are poor substrates for RecAmediated cleavage, more direct studies of repressorRecA binding need to be carried out.
We thank John Reidhaar-Olson for his gift of YC88 dimer, Kendall Knight for his gift of RecA protein, and Rich Breyer for his gift of plasmid pRB103. We also thank Jim Hu for comments on the manuscript. This work was supported by NIH grant AI-16892.
References Ackers, G. K. (1970). A&an. Protein Chem. 24, 343446. Backman, K. C. & Ptashne, M. (1978). Cell, 13, 65-71. Chadwick, P., Hopkins, N., Pirotta, V., Steinberg, R. & Ptashne, M. (1970). Cold Spring Harbor Symp. Quant. Biol. 35, 283-294. Cohen, S.. Knoll, B., Little, J. W. & Mount, I). W. (1981). Nature (London). 294, 182-184. Craig, N. L. & Roberts, J. W. (1980). Nature (London). 283, 26-30. Crowl, R. M., Boyce, R. P. & Echols, H. (1981). .I. Mol. Biol. 152, 815-819. Dreyfus, G., Adam, S. A. & Choi, I). Y. (1984). Mol. Cell. Biol. 4, 415423. Gimble, F. S. (1987). Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, MA. Gimble, F. S. & Sauer, R. T. (1985). J. Bucteriol. 162, 147-154. Gimble, F. S. & Sauer, R. T. (1986). J. Mol. Biol. 192, 3947. Hecht. M. H.. Nelson, H. C. M. & Sauer, R. T. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 26762680. Hecht, M. H., Sturtevant, J. M. & Sauer, R. T. (1986). Proteins: Struct. Funct. Genet. 1. 4346. Horiuchi, ‘i’. & Inokuchi, H. (1967). J. Mol. Biol. 27, 217-224. Ito, K., Date, T. & Wickner, W. (1980). J. Biol. Chem. 255, 2123-2130. Johnson, A., Pabo, C. 0. & Sauer, R. T. (1980). Methods Enzymol. 65, 839-856. Little,
1 Repressor
Shortle, D. (1986). J. Cell. Eiochem. 30, 281-289. Shortle, D. & Lin, B. (1985). Genetics, 110, 53h555. Slilaty, S. N. & Little, J. W. (1987). Proc. Nat. Auzd. Sci., U.S.A. 84, 3987-3991. Slilaty, S. N., Rupley, J. A. & Little, J. W. (1986). Biochemistry, 25, 686G-6875.
Edited
Cleavage
39
Weiss, M. A., Pabo, C. O., Karplus, M. & Sauer, R. T. (1987). Biochemistry, 26, 897-904. Zagursky, R. J. & Berman, M. L. (1984). Gene, 27, 183191.
by M. Gottesman
h Repressor Mutants that are Better Substrates for RecA-mediated Cleavage Frederick S. Gimblet and Robert T. Sauer Department of Biology Massachusetts Institute of Technology Cambridge, MA 02139, U.S.A. (Received 21 January 1988, and in revised form 8 August 1988) RecA-mediated cleavage of the bacteriophage 1 repressor results in inactivation of the protein and leads to induction of the 1 prophage. Here, we report the identifieation of three mutations in 1 repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the in& mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.
1. Introduction An important property of many transcriptional repressors is their ability to be inactivated in response to specific environmental signals. For example, when bacteriophage 1 lysogens are treated with DNA damaging agents, d repressor is cleaved in a RecA-dependent reaction and the prophage is induced (Roberts t Roberts, 1975; Roberts & Devoret, 1983). This cleavage reaction can be reproduced in vitro by incubating purified 1 repressor with a complex of RecA protein, singlestranded DNA, and ATP or an ATP analogue (Roberts et al., 1978; Craig & Roberts, 1980). The single-stranded DNA in the reaction is thought to mimic the inducing signal produced irk vivo by DNA damaging treatments. Although 1 repressor cleavage under physiological conditions is strictly dependent upon RecA protein, repressor cleavage at alkaline pH has been shown to be autocatalytic (Little, 1984). For these reasons, it is thought that RecA protein acts by stimulating the inherent autodigestion activity of I repressor. 3, repressor mutations that prevent prophage induction (ind- mutations) can be divided into two
groups based upon the properties of the corresponding mutant repressors (Gimble t Sauer, 1986). The first group includes mutant repressor proteins that are resistant to RecA-mediated cleavage and resistant to autodigestion at alkaline pH. Some of these group I mutations alter the residues bordering the site at which RecAdependent cleavage and autodigestion occur (Sauer et al., 1982; Little, 1984). The second group of indrepressors are also resistant to RecA-mediated cleavage but these repressors are able to autodigest at alkaline pH. We have argued that these group II mutations identify side-chains that are involved in binding of I repressor to the RecA protein (Gimble & Sauer, 1986). Here, we describe mutations that act as intragenic, second-site suppressors of a group II ind- mutation. Phages containing the ind- mutations and one of the second-site mutations are inducible in vivo and the corresponding revertant repressors are sensitive to RecA-mediated cleavage in vitro. Purified repressors bearing only the secondsite changes have properties similar to the previously described i&‘-l repressor (Cohen et al., 1981); i.e. these repressors are hypersensitive to RecAmediated cleavage and display a reduced ability to form dimers. We show that this reduced dimerization
t Present address: Department of Biochemistry, University of California, Berkeley, Berkeley, CA 94720, U.S.A. 0022-2$3ti/s9/050029-11
$03.00/O
is the
mechanism
changes improve
by
which
the efficiency
the
second-site
of RecA-mediated
cleavage.
29
0 1989 Academic Press Limited
30
F. A’. C&n&
2. Materials and Methods (a) Buffers Standard buffer (SB200) contains 10 miv-Tris HCl (pH 8), 0.1 mM-EDTA, 200 miw-ECl, 1.4 miw-2-mercaptoethanol, 5% (v/v) glycerol (SB50 has 50 m&r-ECl, SB800 has 800 mM-ECl). Lysis buffer is 100 mM-Tris. HCl 200 mM-ECl, 1 mM-EDTA. 2 mM-CaCl,, (PH 8) 1.4 m&r-2-mercaptoethanol. 5% (v/v) 10 mM-MgCl,, glycerol. RecA cleavage buffer contains 50 m&r-ECl, 15 mw-Tris. HCl (pH 7.5), 2 m&r-MgCl,, 2 m&r-dithiothreitol, 0.1 mM-EDTA. Assay buffer is RecA cleavage buffer supplemented with 100 pg bovine serum albumin/ml. (b) Isolation
of revertant
bacteriophages
Revertants of the in& bacteriophage 2 c1 GR185 (Gimble & Sauer, 1985) were isolated following ultraviolet light induction of a mutagenized population of lysogens. Hydroxylamine mutagenesis of Escherichia coli strain GW5100 (1 c1 GR185) was performed according to the procedure described by Miller (1972). Mutagenized cells were grown to logarithmic phase in LB broth, washed once, and resuspended in 5 ml of 0.01 M-MgSG,. Prophage induction was initiated by irradiating a 20-fold dilution (in 091 M-MgSG,) of this culture with (30 J/m’) ultraviolet light. A portion of the cells (1 ml) was diluted X-fold into LB broth, incubated at 37°C for 2 h, and killed by chloroform lysis. Part of this culture (0.1 ml) was plated with 0.2 ml of strain US3 (thi-, his-, la&-, ladYi, supa, recA - ) on Trypticase (BBL Microbiology Systems) plates and phage were allowed to grow overnight at 37°C. Turbid plaques were picked and spotted onto lawns of strain US3 and strain DM1187 (F-. his-4, recA441, s&All, lezA3-51; Mount, 1976). The increased level of activated RecA protein in strain DM1187 results in a clear plaque phenotype for ind+ pseudorevertants or wild-type phage and a turbid plaque phenotype for ind- phage (Mount, 1976). Candidates from 3 independent, mutagenesis experiments, which yielded clear plaques on strain DM1187 and turbid plaques on strain US3, were isolated and plaque-purified twice on strain DM1187. (c) Construction revertant
of plasmids subditutions
bearing
Restriction digestions, ligations and gel electrophoresis were performed by standard procedures (Maniatis et al., 1982). The restriction fragments used for cloning were purified by electrophoresis on low melting point agarose gels (NuSieve, FMC Corp.) or were obtained by electroelution from polyacrylamide gels. The c1 (repressor) gene from the revertant 1 phages was initially cloned into plasmid vector pZ152 (Zagursky & Berman, 1984). Purified 1 DNA from each of the revertants was digested with EcoRI and PvuI and ligated to a preparation of EcoRI and PvuI-cut pZ152. After transformation into E. coli strain X90 (P’2acIq’ pro, ara-, A(Zac-pro)l3, n&A, argEam, thi-, rif), colonies resistant to tetracycline (10 pg/ml) and to infection by ICI- phage were selected. The presence of the c1 gene in candidate plasmids was confirmed by restriction analysis. A 1.1 kbt fragment bearing the c1 gene was purified from a BglII and Pet1 digest of these plasmids and was cloned into t Abbreviations polyethyleneimine.
used: kb, lo3 base-pairs; PEI,
and R. T. Sauer M13mp8 for “dideoxy” sequence analysis @anger pf a/.. 1977). DNA sequencing revealed that each of the reverted cl genes contained the original Gly185+Arg (GRlS5) indmutation, a Leu64-+Met (LMM) mutation, and one of 3 revertant substitutions (Met87-+Thr. MT87: Alal52+Thr, AT152; or Prol58-+Thr. PT158). Wc assumed that the LM64 mutation was silent,: and indeed. found that it had been overlooked in the original sequencing of the GR185 ind- mutant. Subcloning and/or cassette mutagenesis were used to remove the LM64 mutation and to construct overproducing plasmids containing c1 genes with: (1) the GR185 ind- mutation alone; (2) the GR185/MT87, GR185/AT152, or GR185/PT158 double mutations; and (3) the MT87. AT152 or PT158 mutations alone. To confirm thr presence of the proper mutations in these plasmids. the entire CT gene was sequenced. The structure of each plasmid is identical and contains a 164 kb EcoRI -0laI fragment carrying a P,,,-i repressor fusion (Backman & Ptashne, 1978) cloned between the EcoRI and C:ZaI sites of plasmid pZ150 (Zagursky & Berman, 1984). Plasmids pFG610 and pFG611 are P,,, derivatives of the MT87 and PT158 plasmids described above. (d) Inactivation
of mutanf
repressors
in vivo
The sensitivity to RecA-mediated inactivation of mutant repressors borne on plasmids was assayed by phage spot tests in the presence or absence of 0.7 p’g mitomycin C/ml (Gimble & Sauer, 1985). (e) Protein purijcations The wild-type and mutant repressor proteins were purified essentially as described by Johnson et al. (1980). Repressor proteins labeled with [35S]methionine were purified using a modification of this method. Strain X90 containing plasmids pMH236 (encoding wild-type repressor; Hecht et al., 1986) pFG610, or pFG611 was used for the purifications. Cells were grown in M9 minimal medium (100 ml) containing ampicillin (100 pg/ml), arginine (40 pg/ml) and vitamin Bl (1 pg/ml) (Miller, 1972) at 37°C to an approximate concentration of 5 x 10s cells/ml. The culture was induced by the addition of 100 pg isopropyl-1-thio-/I-n-galactoside/ml, grown for an additional 20 min and then labeled with [35S]methionine (5 mCi, 1000 Ci/mmol; New England Nuclear) for 5 min. After the cells were harvested by centrifugation, they were resuspended in lysis buffer to a concentration of 10 mg/ml, lysed by sonication, and centrifuged at 4°C for 1 h at 14,000g. The supernatant was decanted and nucleic acids and cell debris were precipitated by adding polyethyleneimine (PEI) to 0.20/, (v/v) followed, by centrifugation. Two volumes of a saturated solution of (NH&SO, were added to the PEI supernatant, the solution was stirred overnight, and the protein pellet was collected by centrifugation at 16,000g for 1 h. The pellet was resuspended in 16 ml SB50 buffer/g of cells, and the solution was dialyzed extensively against SB50. The dialysate was loaded onto a 1 cm x 8 cm column of CMSephadex (C-50; Pharmacia Fine Chemicals) for wild-type and PT158 repressors, or onto a 1 cm x 8 cm column of Affi-gel blue (Bio-Rad) for the MT87 protein. The columns were washed with SB50 and repressor was eluted with SB300 for the CM-Sephadex columns and SB800 for the Affi-gel blue column. Fractions containing labeled repressor were pooled and loaded onto a 1 cm x 8 cm column of hydroxylapatite (Clarkson Chemical Co.) that
1 Repressor
had been equilibrated in 0.2 M-potassium phosphate (pH 8.0). The columns were washed with the same buffer and repressor wm eluted using a single step of 1.0 M-potassium phosphate (pH 8-O). The fractions containing repressor were pooled, concentrated by ultrafiltration (YM-10 membrane; Amicon Corporation), and then dialyzed against RecA cleavage buffer. The specific radioactivities of these repressors were estimated to be 2 x 1016 cts/min per mol by scintillation counting and SDS/polyacrylamide gel electrophoresis. The disulfide-bonded YC88 dimeric repressor and the YC88 repressor, which had been reduced and alkylated with iodoacetamide, were gifts from John ReidhaarOlson. RecA protein and the IS84 repressor were gifts from Dr Kendall Knight and Kathy Hehir, respectively. The C-terminal 93-236 repressor fragment was purified from strain X90 bearing plasmid pFG401. This plasmid encodes i repressor residues 93 to 236 under transcriptional control of a tat promoter (Gimble, 1987). The 93-236 fragment was purified by ion exchange chromatography on QAE-Sephadex and DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose, and gel chromatography using Ultrogel AcA44. filtration Sequential Edman degradation confirmed that the purified fragment had the N-terminal sequence Met-GlnPro-Ser-Leu expected for a fragment starting at residue 93 of int,art repressor (Gimble, 1987). ( f) RecA-mediated cleavage and autodigrstion RecA-mediated cleavage of wild-type or mutant repressors was generally assayed by incubating samples containing 4.5 PM-repressor, 2.7 q-Red protein, 1 miv-adenosine 5’-[y-thio]triphosphate (BoehringerMannheim), 4.7 pg poly(dT)/ml (PL Biochemicals), in RecA cleavage buffer at 37°C. Portions of the samples (30 ~1) were withdrawn at several time points and the reactions were stopped by the addition of 10 ~1 of SDS sample buffer and heating to 90°C. When RecA-mediated cleavage of the YC88 dimer was analyzed, reducing agent was omitted from the reaction mixtures and from the SDS sample buffer, and 1 ~1 of a 1 M solution of iodoacetamide was added to each reaction immediately prior to addition of SDS sample buffer to block sulfhydryl-disulfide rearrangements. Autodigestion reactions were performed using a modification of the methods described by Little (1984). Samples containing 3.2 PM-repressor, 50 mM-KCl, and 50 mM-CAPS. NaOH (pH 10.5) were incubated at 37°C. and portions (30~1) were removed at various times. The reactions were stopped by the addition of 10 ~1 of SDS sample buffer and by heating. The extent of cleavage in most autodigest,ion and RecA-mediated cleavage experiments was determined by running samples on SDS/ urea/l 80,;) polyacrylamide gels (Ito et al.. 1980) and staining with Coomassie brilliant blue. Stained gels were scanned using au LKB 2202 ultroscan laser densitometer equipped with an LKB 2220 recording integrator. To quantify the extent of cleavage, the peak areas of the 2 digestion fragments were added and calculated as a percentage of the total repressor area, which is the sum of the peak areas of the fragments and of the remaining intact repressor. Rates of RecA-mediated cleavage of wild-type or mutant repressors at different total repressor concentrations were determined using a modification of the procedure of Cohen et aZ. (1981). Samples containing 5 nm-[“5S]repressor, 1 rnM-adenosine 5’.[y-thioltriphosphate. 100 pg bovine serum albumin/ml in RecA
Cleavage
31
cleavage buffer were incubated at 4°C for 30 min in the presence or absence of 1 PM-unlabeled repressor of the same type. After the solution was heated for 5 min at 37°C. it was combined with a solution of 54pm-RecA protein and 9.3 pg poly(dT)/ml, which had been preincubated for 30 min. Portions (30 ~1) of each reaction mixture were withdrawn at different times, the reactions were stopped by heating in SDS sample buffer, and the samples were electrophoresed on 15y0 protein gels (Dreyfus et aZ., 1984). To visualize the repressor and fragment, bands, small amounts of unlabeled repressor or cleavage fragments were added to each mixture prior to electrophoresis. After the gels were stained and destained, the bands containing intact repressor and the 2 fragments were excised, placed into different scintillation vials containing 9 ml of Soluscint-0 (National Diagnostics), 1 ml of Hydrofluor (National Diagnostics) and 0.5 ml Solusol (National Diagnostics), heated at 50°C for 2.5 h, and then left, overnight before counting. The degree of cleavage was calculated as the ratio of the radioactivity in the 2 fragment bands to the radioactivit,? in the 2 fragment bands and the repressor band. (g) Nitrocellulose Jilter binding of operator DNA experiments Nitrocellulose filter-binding were performed in assay buffer using the general methods described by Gimble & Sauer (1986). A restriction fragment containing the lORl site was used as the source of operator DNA (Gimble & Sauer, 1986). Experiments were done in triplicate, and the repressor concentrations at half-maximal binding were calculated by computer curve-fitting of the data using linear regression. (h) Gel filtration
chromatography
The apparent molecular weights of repressor oligomers were determined by gel filtration chromatography. Samples (200 pl) containing repressor at a concentration of 4 pM and a myoglobin standard were injected onto a 0.75 cm x 60 cm Bio-Sil TSK-125 HPLC gel filtration column (Bio-Rad Laboratories) and were chromatographed in a buffer containing 15 mill-Tris. HCI (pH 7.5), 50 mM-KCl, 0.1 mM-EDTA, 2 mM-M$l,. All experiments were performed at 37 “C. Protein peaks were detected by ultraviolet absorbance at 280 nm. The columns were calibrated with bovine serum albumin, ovalbumin, carbonic anhydrase. myoglobin and vitamin B12.
3. Results (a) Isolation and sequence analysis of revertants A I prophage bearing an ind- mutation cannot be induced because its mutant repressor cannot be inactivated by RecA-mediated cleavage (Roberts t Roberts, 1975). As a consequence, revertants can be isolated by inducing a mutagenized culture of an indlysogen with ultraviolet irradiation and screening among the small number of progeny phage for those having an ind+ phenotype. We used a selection and screening method of this type to isolate three independent revertants of the indphage Iz CT GR185 (see Materials and Methods). The revertant repressor genes were cloned into plasmids, and mutations were located by DNA sequencing.
3%
F. S. Nimble and R. T. Sau,er
Table 1
Table 2
Second site mutations that restore an ind+ phenotype to th,e GR185 repressor Code
Second-site mutation
MT87
Met87 +Thr ATG ACG Ala152 +Thr GCA ACA Pro158 +Thr ACA I, CCA
AT152 PT158
of mutant repressors in vivo
Indu,cibility
Each revertant retained the original ind- mutation (GR185) and a silent mutation (LM64) that was present in the parental ind- phage. In addition, each revertant acquired a second-site mutation (MT87, AT152, or PT158; see Table 1). To avoid complications in characterizing the revertants, we constructed overproducing plasmids containing repressor genes without the silent mutation. We also constructed repressor genes in which the second-site mutations were present in otherwise wild-type backgrounds. (b) RecA-mediated cleavage properties of the mutants in vivo The sensitivity to RecA-mediated cleavage in ~ivo of the revertant repressors and repressors bearing only the second-site mutations was assayed by phage spot tests in the presence and absence of the inducing agent mitomycin C (see Table 2). In the absence of induction, each of the plasmid-borne repressors listed in Table 2 prevents lytic growth of the infecting phage. This indicates that each repressor is able to bind to operator DNA. The strain containing the GR185 ind- repressor is as resistant to phage infection under inducing conditions as under non-inducing conditions. As noted
Spot phenotype ( -MC) Induced ( + MC)
Uninduced
Allele
(‘Iear Turbid Turbid Turbid
No reprevsor Wild-type GR185 GR185/MT87 GR185/AT152 GR185/PT158 MT87 AT152 PTl58
‘hbid
(:lear Semi-turbid Turbid Semi-turbid Semi-turbid &m-turbid
Turbid Turbid Turbid
( kar (Year
Turbid
(:ll%L:
For details of the assay, see Gimble & Sauer (1985). +MC, 0.7 pg mitomycin C/ml.
earlier, this results from the resistance of the indrepressor to RecA-mediated cleavage (Gimble & Sauer, 1985). Strains containing wild-type repressor and the revertant repressors (GR185/AT152, GR185/PT158, GR185/MT87) are somewhat sensitive to phage infection under inducing conditions. This suggests that these repressors can undergo RecA-dependent cleavage in vivo. Finally, in the presence of mitomycin C, strains containing repressors that bear only the second-site mutations (MT87, AT152, PT158) behave like strains that contain no repressor; i.e. they are extremely sensitive to phage infection. This result suggests that repressors bearing only the second-site changes are more sensitive to RecA-mediated cleavage in vivo than wild-type repressor. (c) RecA-mediated mutant
cleavage of puri$ed. repressors
Wild-type repressor, the GR185 ind- repressor, revertant repressors bearing the GRI 85/MT87,
Wild type
GRl85
20
i-----J-t 20 240 0
PTl58
GRl85lPTl58
---II
L 0
240
J 0
20
240
0
20
240
Time (min)
Figure 1. Analysis of RecA-mediated cleavage of mutant and wild-type repressor proteins by SDS/polyacrylamide gel electrophoresis. The extent of RecA-mediated cleavage for the wild-type, GR185, GRlf%/P’I’158, and PT158 repressors is shown after 0, 20, and 240 min of incubation at 37°C. Intact repressor is indicated as IR, and the products of digestion are indicated as irl (residues 112 to 236) and dr2 (residues 1 to 111).
A Repressor Cleavage
repressor monomers (R) relative to dimers (R,) by changing the dimerization constant K, . According to the model, only the repressor monomer binds to and is cleaved by RecA (Phizicky & Roberts, 1980) and an increase in the monomer concentration would increase the observed rate of RecA-mediated cleavage as long as RecA was not saturated. As we discuss below, the suppressor mutations decrease the ability of repressor to form dimers. Mutations could also increase the rate of RecA-mediated cleavage by increasing the rate of turnover of the RecA-R complex (increasing /&) or by increasing the affinity of the repressor monomer for RecA (decreasing the dissociation constant, KS).
GRl&/AT152 and GRl&/PT158 changes, and repressors bearing the single MT87, AT152 and PT158 changes were purified and assayed for sensitivity to RecA-mediated cleavage in vitro. As shown in Figure 1, RecA-mediated cleavage of the GR185 repressor is extremely slow and only trace amounts of cleavage products are seen after four hours of digestion. By contrast, wild-type repressor, the revertant repressors, and repressors containing only the second-site mutations are cleaved at much faster rates. Figure 2 shows time-courses of RecAmediated digestion for each of the repressor proteins. The GRl85/AT152 and GRl85/MT87 mutants are digested somewhat more slowly than wild-type, whereas the GR185/PT158 repressor is digested slightly faster than wild-type. Strikingly, the MT87, AT152 and PT158 repressors are digested at rates sixfold to Sl-fold faster than the wild-type protein under these conditions (Table 3). The relative order of cleavage rates is the same (PT158>AT152>MT87) whether the ind- mutation is present or absent, although cleavage of each protein is about 20-fold to 30-fold faster in the absence of the in& substitution. There are three simple ways in which the suppressor mutations might increase the rate of RecA-mediated cleavage of repressor. These can be discussed in terms of the following model for RecAmediated cleavage: KS k RecA + R o RecA: R * RecA + fragments
A
li Kl 0 R2 mutation
could
increase
the
(d) Autodigestion of the mutant repressors The repressor autodigestion reaction at high pH and the RecA-mediated cleavage reaction are thought to share a common catalytic mechanism (Little, 1984, Slilaty et al., 1986; Gimble & Sauer, 1986). Thus, the rate of autodigestion should be related to the rate of turnover of the RecA-R complex. To test for possible effects of the suppressor mutations on the catalytic rate constant (lcJ, we examined the rates of autodigestion for the mutant repressors. Table 3 shows that the revertants and the repressors bearing the suppressor mutations autodigest at rates that are very similar to the wild-type rate, Hence, the revertant substitutions do not appear to increase the rate of RecA-mediated cleavage by increasing the catalytic rate constant. It should be noted, however, that this conclusion could be incorrect if the detailed mechanisms of cleavage are different for autodigestion and RecA-mediated cleavage.
(1)
population
33
of
too
- 7
/
/
MT07
PTl58lGRl85.
y
80
120
ATl52/GR185,
180
240
Time (mid
Figure 2. Kinetics of RecA-mediated cleavage of mutant and wild-type repressors. The purified proteins were digested for different times at 37”C, and the extent of cleavage at each time point was measured by SDS/polyacrylamide gel electrophoresis as described in Materials and Methods.
F. S. Nimble and R. T. Sauer
34
Table 3 Ret A -mediated cleavage and autodigestion RecA-mediated cleavage? (relative rate)
Allele
Autodigestion$ (relative rate)
la
l@§
Wild-type GR185 GR185/MT87 GR185/AT152 GR185/PT158 MT87 AT152 PT158
< 0.05 0.2 0.4 1.6 6.0 9.2 31.1
1.1 0.9 0.9 0.8 0.9 0.8 0.9
15.0 8.0
n.d. n.d.
IS84 93236 fragment
n.d., not determined. t Reaction mixtures contained 4.5 par-repressor, 2.7 pa-RecA protein, 1 mM-adenosine 5’-[y-thioltriphosphate, and 4.7 pg poly(dT)/ml in RecA cleavage buffer. $ Reaction mixtures contained 3.2 fin-repressor, 50 mM-KCl, and 50 mM-CAPS NaOH (pH 10.5) and were incubated at 37°C. Q The initial rate of RecA-mediated digestion of wild-type repressor is 39 ng/min per pg RecA protein. 11The wild-type rate constant for autodigestion is 166 x 10-a 6-i.
(e) Dimerization of the mutant repressors To study repressor dimerization, gel filtration chromatography was used to compare the elution positions of wild-type repressor and the MT87, AT152 and PT158 repressors (Table 4). Because monomers and dimers of repressor are in rapid equilibrium in such experiments (Sauer, 1979), the proteins elute as trailing peaks and a rigorous analysis of the monomerdimer equilibrium is not if each possible (Ackers, 1970). Nevertheless, repressor is loaded at the same initial concentration and is chromatographed under the same conditions, then it should be possible to judge the relative ability of each of the repressors to dimerize. Under the chromatographic conditions of these experiments, wild-type repressor elutes at an apparent molecular weight of 41,000. This value is intermediate between those expected for a monomer (26,000) and a dimer (52,000). The MT87, AT152 and PT158 proteins elute at apparent molecular weights that are from 4000 to 8000 less than the apparent molecular weight at which wild-type repressor elutes (Table 4). Thus, each of these
mutations appears to impair the ability of repressor to dimerize. Because repressor dimers are the oligomeric species that bind to operator DNA (Chadwick et al., 1970; Sauer, 1979), a mutant displaying reduced dimerization should also display reduced operator binding activity. This is clearly the case for the AT152 and PT158 repressors, which have approximately l/7 of the wild-type binding activity as assayed by nitrocellulose filter binding (Table 4). If these mutations affect DNA binding only by reducing dimerization, then the observed reduction in DNA binding activity would indicate an approximate 50-fold increase in the equilibrium dissociation constants for dimerization of the AT152 and PT158 repressors. The MT87 repressor shows no operator binding activity in the assay used (Table 4), but this result is difficult to interpret as other mutant repressors bearing N-terminal substitutions have been shown to have operator binding activity which is not detected by the filter binding assay (Nelson & Sauer, 1986). In fact, the MT87 mutant displays approximately the same operator binding activity in Z&JOas the AT152 and
Table 4 Effects on dimerization Allele Wild-type MT87 AT152 PT158
and operator binding
Apparent molecular weight
Relative operator binding activity?
41,000 37,000 37,000 33,000
1.0 Not detected 0.14 0.14
t Defined ae the concentration of wild-type repressor required to give half-maximal operator binding divided by the concentration required to give half-maximal operator binding for the repressor tested. Assays were performed by the nitrocellulose filter aaaay as described in Materials and Methods.
1 Repressor
100
(a) G
”
--
,n-Q hi
,oow
, Wild +w ,
PT158
35
Cleavage
The model also predicts that the concentration dependence of the relative cleavage rate should be reduced for mutants that dimerize less well than wild-type. Figure 3 shows experiments of this type for high (1 PM) and low (5 nM) concentrations of wild-type repressor, the PT158 repressor and the MT87 repressor. These experiments were performed with total RecA protein in excess over total repressor to avoid complications caused by saturation of RecA protein. For wild-type repressor, the relative rate of cleavage is about fourfold faster at the low than at the high concentration. The relative rate of cleavage of the MT87 repressor is about twofold faster at the low concentration. and that of the PT158 repressor is the same at the low and high concentrations. These data are therefore consistent with the model and the idea that the MT87 and PT158 mutations reduce the ability of repressor to dimerize. The dimerization constant for wild-type repressor is approximately 20 nM (Sauer, 1979) and, in the absence of RecA protein, approximately lo?/, of the repressor should be monomeric at a concentration of 1 pM and 70% should be monomeric at a concentration
I 60
I 30 Time
I ‘90
(min)
of 5 nM. Thus, a sevenfold
difference in the relative initial rates at’ the low and high concentrations would be expected if the monomer-dimer equilibrium were not perturbed by RecA binding. The observed fourfold differences in rates would be expected if the RecA-repressor monomer affinity constant (K,) were approximately 3 PM. This value should be viewed as an extremely rough estimate, however, as the data are somewhat uncertain and the calculation depends upon several assumptions.
Figure 3. Kinetics of RecA-mediated cleavage of mutant and wild-type repressors at 2 different repressor concentrations. RecA-mediated cleavage of purified proteins was carried out at 37 “C as described in Materials and Methods using initial repressor concentrations of 5 mu (closed symbols) and 1 PM (open symbols). In these experiments, the concentration of RecA protein was repressor; (b) PT158 repressor: 5.4 PM. (a) Wild-type (c) MT87 repressor.
PT158 mutants, suggesting that it has binding activity in vitro which is not detected by the filter binding assay. Overall, the gel filtration and operator binding experiments are consistent and indicate that the MT87, AT152 and PT158 mutations interfere with repressor dimerization. (f) Effects of repressor concentration on RecA-mediated cleavage
The model of equation (1) predicts that the relative rate of RecA-mediated cleavage of wildtype repressor should be faster at low concentrations, where repressor is predominantly monomeric, than at high concentrations, where repressor is predominantly dimeric (Phizicky & Roberts, 1980).
(g) Additional
tests of the model
(i) Cleavage of other mutants reduced dimerization
displaying
If repressor dimers cannot be cleaved by RecA (model of eqn (l)), then most mutants displaying reduced dimerization should also display increased rates of RecA-mediated cleavage relative to wildtype. To test this prediction, we used two repressors bearing mutations that were not selected as revertants of an ind- phenotype. The first mutant contains the IS84 (Ile84+Ser) missense mutation. IS84 was originally isolated as a mutant defective in DNA binding and the protein was subsequently shown to be defective in dimerization (Hecht et al., 1983; Weiss et al., 1987). The second mutant contains repressor residues 93 to 236 but is deleted for the N-terminal domain of repressor. The 93-236 fragment dimerizes less efficiently than wild-type repressor because it is missing the N-terminal dimerization contacts (Pabo & Lewis, 1982; see Discussion). The data of Figure 4 and Table 3 show that the IS84 mutant and the C-terminal 93-236 fragment undergo RecA-mediated digestion considerably faster than the wild-type repressor and somewhat
F. 8. Gimble and R. T. Sau.rr
36
0
RecA
b
YC88 dwner
Wild type -r-----Ii.----c d
YC88 monomer
e
f
g
h
0
240
0
240
* ,:* 2’ I
Rearessor
Repressor fraqments r
--II 0
240
Time (min) 30 Time (min)
Figure 4. Kinetics of RecA-mediated cleavage of the 93-236 repressor fragment, the IS84 and MT87 repressors, and wild-type repressor. The purified proteins were digested at 37°C for different times as described in Materials and Methods.
faster than the MT87 repressor. Therefore, repressor mutants displaying reduced dimerization are more sensitive to RecA-mediated cleavage even in cases where the mutation was not selected on the basis of reverting an ind- phenotype.
Figure 5. Analysis of RecA-mediated cleavage of wildtype repressor, the disulfide-bonded YC88 dimer, and reduced and alkylated YC88 by SDS/polyacrylamide gel electrophoresis. The extent of RecA-mediated cleavage after incubation at 37°C for 0 or 240 min is shown for wild-type repressor (lanes c’ and d); disulfidr-bonded YC88 dimer (lanes e and f); and reduced and alkylated YC88 (lanes g and h). Lanes a and b show the wild-type repressor and the disulfide-bonded YC88 dimer, respectively, in the absence of RecA protein. Alkylation of Cys88 causes repressor and its digestion products to migrate more slowly than the analogous unmodified repressor and fragments (compare lanes d and h). A small amount of unalkylated C-terminal fragment is observed in lane h.
(ii) Cleavage of a covalent repressor dimer According to the model represented in equation (1 ), a repressor that is always dimeric shotild not undergo RecA-mediated cleavage. We have tested this prediction by using a repressor that contains a substitution (Tyr8GCys: YC88) that permits a disulfide bond to form between the two monomer subunits (Pabo & Suchanek, 1986; Sauer et al., 1986). Figure 5 shows that after incubation with RecA for four hours, half of the wild-type repressor has been cleaved but no detectable cleavage of the disulfide-bonded YC88 dimer has occurred (compare lanes d and f). Thus, the covalent dimer is extremely resistant to RecA-mediated cleavage. It is unlikely that this resistance is caused by any gross conformational change in the protein because the disulfide-bonded YC88 dimer binds strongly to operator DNA (J. Reidhaar-Olson, personal communication) and underdoes autodigestion at the same rate as wild-type repressor (data not shown). To control for the possibility that the YC88 mutation interferes directly with RecA-mediated cleavage, we reduced the disulfide bond and alkylated Cys88 with iodoacetamide prior to incubation with the RecA protein. This modified YC88 protein is sensitive to RecA-mediated cleav-
age (Fig. 5, lane h) and, in fact, is cleaved faster than wild-type repressor (compare lanes d and h). The faster cleavage of the alkylated YC88 protein probably results from reduced dimerization, as residue 88 forms part of the dimer interface of the N-terminal domain of repressor (Pabo & Lewis, 1982; see Discussion).
4. Discussion We isolated three second-site mutations that suppress the effects of a group II ind- mutation in 1 repressor. In the cell, the indrepressor is insensitive t,o RecA-mediated cleavage, revertant repressors containing the indmutation and a suppressor mutation are sensitive to cleavage, and repressors containing only the suppressor mutations are hypersensitive to cleavage. Similar phenotypes are observed in studies using purified RecA and repressor proteins. The indrepressor is not detectably cleaved in the RecA-mediated reaction in vitro, repressors bearing the ind- mutation and a suppressor mutation are cleaved at rates similar to wild-type, and repressors bearing only the
II Repressor
suppressor mutations are cleaved at rates significantly faster than wild-type. Thus, reversion analysis has allowed us to identify residue changes that make 1 repressor a better substrate for RecAmediated cleavage. Similar strategies of intragenic reversion have been used to identify sequence changes that enhance the affinity and specificity of 1 repressor-operator binding (Nelson & Sauer, 1985) and sequence changes that enhance the thermodynamic stability of staphylococcal nuclease (Shortle & Lin, 1985; Shortle, 1986). How do the suppressor mutations increase the rate of RecA-mediated cleavage of 1 repressor’! We believe that the main effect of the MT87, AT152 and PT158 mutations is to interfere with repressor dimerization. At concentrations where wild-type dimers normally predominate (above 20 nM; Sauer, 1979), each of the suppressor mutations causes an increase in the fraction of repressor present as monomer. By hypothesis, this should increase the rate of cleavage because the repressor monomer is the true substrate in the RecA-mediated reaction. Phizicky & Roberts (1980) originally proposed that the repressor monomer was the preferred substrate for RecA-mediated cleavage and our results provide strong support for their idea. First, each of the repressors containing a suppressor mutation alone displays reduced dimerization and undergoes RecAmediated cleavage faster than the wild-type repressor. Second, repressor mutants which display reduced dimerization but which were not selected on the basis of an Ind phenotype also exhibit enhanced rates of RecA-mediated cleavage. Third, a disulfide-linked repressor dimer is not cleaved in the RecA-mediated reaction even though it undergoes autodigest.ion at a rate indistinguishable from wildtype. Do the suppressor mutations affect RecAmediated cleavage solely by affecting repressor dimerization? We cannot answer this question rigorously because we have not determined equilibrium dimerization constants for the MT87, AT152 and PT158 mutants or kinetic parameters (K,,, and V,,,) for RecA-mediated cleavage of these repressors. However, at low concentrations where most repressor molecules should be monomeric, the MT87 and PT158 repressors are cleaved only slightly faster than wild-type repressor (Fig. 3). This indicates that these mutations act mainly by affecting repressor dimerization, and allows us to argue that the PT158 mutation, which is the strongest suppressor, interferes with dimerization to a greater extent than the MT87 mutation. This ranking is consistent with the gel filtration experiments. The MT87 and AT152 changes appear to have approximately the same effect on dimerization as assayed either by RecA-mediated cleavage at high repressor concentrations or gel filtration. The overall ranking from strongest to weakest dimerization would thus appear to be wildtype > MT87 = AT152 > PT158. The operator DNA binding experiments give a somewhat different order, but this may simply indicate that the
Cleavage
37
mutations affect operator binding directly as well as via dimerization. Because the suppressor mutations decrease dimerization, it is reasonable to ask if the original GRl85 itimutation increases repressor dimerization. Previously, we found that the GR185 mutation increased operator binding slightly, consistent with an approximate threefold increase in dimerization (Gimble & Sauer, 1986). We argued, however, that this increase was not sufficient to fully account for the resistance of the mutant to RecA-mediated cleavage and suggested that the GR185 change also affected RecA binding. How good is this argument? On the one hand, it is vulnerable because the change in DNA binding caused by the Gly185+Arg mutation may not accurately reflect changes in dimerization. On the other hand, we have also found that repressors with Glu185 and Va1185 are also ind- (Gimble & Sauer, 1985, 1986; J. Hu 6 R. Sauer, unpublished results). It is difficult to imagine how the side-chains of these three amino acids, which are chemically dissimilar, could all mediate improved repressor dimerization. By contrast, it is easy to envision how each sidechain could interfere with the binding of RecA. Hence, we feel that our original conclusion is probably correct, i.e. the GR185 mutation influences both dimerization and RecA binding. This issue could presumably be addressed directly by studying the RecA-mediated cleavage of the GR185 mutant at low repressor concentrations. The increased RecA-mediated cleavage activity reported here for the repressors bearing the suppressor mutations (MT87, AT152 and PT158) has also been observed in the case of the ind”-1 and iti”-2H mutant repressors (Crow1 et al., 1981; Cohen et al., 1981). Lysogens containing these 1 ind” alleles are hypersensitive to inducing treatments (Horiuchi & Inokuchi, 1987), and the id-1 repressor, like the mutants described here, dimerizes less well than the wild-type repressor (Cohen et al., 1981). The ,ind”-1 mutation is a Glu233+Lys substitution, close to the carboxyl terminus of repressor (Gimble & Sauer, 1985). How might the suppressor mutations affect dimerization? Intact 1 repressor contains two apparently distinct sets of dimerization contacts. The isolated N-terminal domain (residues 1 to 92) forms dimers in the crystal structure and in solution (Pabo & Lewis, 1982; Weiss et al., 1987). These N-terminal dimers are stabilized by hydrophobic helix 5-helix 5’ packing interactions which include the packing of Met87 against Ile84’, and Ile84 against Met87’. The MT87 suppressor mutation (Met87-+Thr), would be expected to reduce the free energy of N-terminal dimerization in two ways. First, the hydrophilic hydroxyl group of Thr87 would be introduced into the hydrophobic interface. Second, the S6 and GE groups of Met87, which contribute directly to the packing interface, would be removed. Thus, the crystal structure of the N-terminal domain allows us to rationalize the reduction in repressor dimerization that’ is caused
by the MetS’I-+Thr substitution. Similar arguments can be advanced to explain the reduced dimerization caused by the Ile84-+Ser mutation. The structure of the C-terminal domain of L repressor is not known, but a C-terminal fragment consisting of residues 132 to 236 forms dimers in solution (Pabo et al., 1979). Because the AT152 (Ala152-+Thr) and PT158 (Prol58+Thr) suppressor mutations are close in the primary sequence, it seems likely that this general region of the C-terminal domain plays an important role in mediating dimerization of the C-terminal domain. The wild-type Ala152 and/or Pro158 side-chains might form part of a hydrophobic dimer interface. In this case, substitution of either residue by threonine would be expected to disrupt dimerization in a manner analogous to that described for the MT87 mutation. Alternatively, the AT152 and/or PTI 58 mutations might cause conformational changes that affect dimerization indirectly. It seems clear that the repressor strongly preferred substrate for
monomer is the RecA-mediated
cleavage but the mechanism by which the dimer is resistant to cleavage is less clear. The simplest model is that RecA cannot bind dimers because the binding surface is occluded or altered by dimerization, but there are no direct experiments that show that RecA is unable to bind the repressor dimer. Alternatively, dimer-RecA complexes might form but, be unable to undergo cleavage. Slilaty et al. (1986) have suggested that repressor dimers do not autocleave, based upon their finding that the rate of autocleavage shows a concentration dependence under certain conditions. This would be consistent with the idea that dimer-RecA complexes might, not undergo cleavage. In fact, two of our mutations that affect dimerization, AT152 and PT158, are close in the primary sequence to Ser149, which is thought to play a catalytic role in the cleavage reactions (Slilaty & Little, 1987). As a result, it is plausible that some of the active site residues could be buried or occluded by repressor dimerization. On the other hand, we have observed that the disulfidebonded YC88 repressor dimer autocleaves at a rate similar to wild-type, which would seem to argue that the active site cannot be inaccessible in the dimer. A problem, here, is that we do not know that the C-terminal domains of the covalent dimer are actually in contact under the conditions (pH 10.5, 37°C) where our autocleavage experiments were performed. This issue needs to be studied further. Moreover, to directly address the issue of why repressor dimers are poor substrates for RecAmediated cleavage, more direct studies of repressorRecA binding need to be carried out.
We thank John Reidhaar-Olson for his gift of YC88 dimer, Kendall Knight for his gift of RecA protein, and Rich Breyer for his gift of plasmid pRB103. We also thank Jim Hu for comments on the manuscript. This work was supported by NIH grant AI-16892.
References Ackers, G. K. (1970). A&an. Protein Chem. 24, 343446. Backman, K. C. & Ptashne, M. (1978). Cell, 13, 65-71. Chadwick, P., Hopkins, N., Pirotta, V., Steinberg, R. & Ptashne, M. (1970). Cold Spring Harbor Symp. Quant. Biol. 35, 283-294. Cohen, S.. Knoll, B., Little, J. W. & Mount, I). W. (1981). Nature (London). 294, 182-184. Craig, N. L. & Roberts, J. W. (1980). Nature (London). 283, 26-30. Crowl, R. M., Boyce, R. P. & Echols, H. (1981). .I. Mol. Biol. 152, 815-819. Dreyfus, G., Adam, S. A. & Choi, I). Y. (1984). Mol. Cell. Biol. 4, 415423. Gimble, F. S. (1987). Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, MA. Gimble, F. S. & Sauer, R. T. (1985). J. Bucteriol. 162, 147-154. Gimble, F. S. & Sauer, R. T. (1986). J. Mol. Biol. 192, 3947. Hecht. M. H.. Nelson, H. C. M. & Sauer, R. T. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 26762680. Hecht, M. H., Sturtevant, J. M. & Sauer, R. T. (1986). Proteins: Struct. Funct. Genet. 1. 4346. Horiuchi, ‘i’. & Inokuchi, H. (1967). J. Mol. Biol. 27, 217-224. Ito, K., Date, T. & Wickner, W. (1980). J. Biol. Chem. 255, 2123-2130. Johnson, A., Pabo, C. 0. & Sauer, R. T. (1980). Methods Enzymol. 65, 839-856. Little,
1 Repressor
Shortle, D. (1986). J. Cell. Eiochem. 30, 281-289. Shortle, D. & Lin, B. (1985). Genetics, 110, 53h555. Slilaty, S. N. & Little, J. W. (1987). Proc. Nat. Auzd. Sci., U.S.A. 84, 3987-3991. Slilaty, S. N., Rupley, J. A. & Little, J. W. (1986). Biochemistry, 25, 686G-6875.
Edited
Cleavage
39
Weiss, M. A., Pabo, C. O., Karplus, M. & Sauer, R. T. (1987). Biochemistry, 26, 897-904. Zagursky, R. J. & Berman, M. L. (1984). Gene, 27, 183191.
by M. Gottesman