183

Mutation Research, 250 (19911 183-197 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100177S

MUT 02513

N e w m u t a t i o n s in c l o n e d Escherichia coli umuDC genes: N o v e l p h e n o t y p e s of strains carrying a umuC125 p l a s m i d Lorraine Marsh h, Takehiko Nohmi c, Sean Hinton a and Graham C. Walker a Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.), b Department of Cell Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461 (U.S.A.) and c Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, Tokyo 158 (Japan) (Accepted 5 April 19911

Keywords: UmuDC genes, New mutations in; UmuC125 plasmid, Novel phenotypes

Summary The u m u D C locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli u m u D C genes cloned on pBR322-derived plasmids were isolated by two methods. First, spontaneously-arising mutant u m u D C plasmids that failed to confer cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant u m u D C plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD+C ÷ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD ÷C ÷ plasmids, pBR322-derived umuD ÷C ÷ plasmids inhibited the induction of the SOS response of lexA ÷ strains as measured by expression of din::Mu dl(lac Ap) fusions but most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid, pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA ÷ strains to killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis, pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA ÷ strains. The effect of pLM109 on the UV sensitivity of/exA(Def) strains was similar to that of the parental umuD ÷C ÷ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G : C ~ A : T transition that changed codon 39 of umuC from GCC --, GTC thus changing Ala39 to Va139. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.

Most mutagenesis by ultraviolet radiation and many chemicals in Escherichia coli requires the products of the u m u D C operon (for reviews see

Correspondence: Dr. Graham C. Walker, 56-621, Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.).

Witkin, 1976; Walker, 1984, 1985; Peterson et at., 1988). Mutations in either umuD or umuC virtually completely block mutagenesis after UVirradiation and make strains somewhat more sensitive to the killing effects of UV (Kato and Shinoura, 1977; Steinborn, 1978; Elledge and Walker, 1983a; Shinagawa et at., 1983). The plasmid pKM101 carries an evolutionarily diverged

184 analog of the u m u D C operon, mucAB, that can substitute for its role in mutagenesis (Walker and Dobson, 1979; Perry, et at., 1982, 1985). "l'hc u m u l ) C operon is regulated as part of the SOS response of E. coil: it is repressed by the LexA protein and its expression increases upon SOS-induced RecA-mediated cleavage of LexA (Bagg ct al.. 1981: Shinagawa et al., 1983, Walker, 1985). In addition, U m u D undcrgocs a RecA-mediatcd cleavage (Burckhardt el al., 1988; Shinagawa c! at., 1988) that posttranslationally activates it for its role in mutagenesis (Nohmi et al., 1988). In addition, recent evidence has suggested that mutagenesis by UV and various chemicals rcquircs an additional function of the RecA protcin besides thc clcavagc of LexA and U m u D (Nohmi ct al., 1988; Dutreix et al.. 1989: Ennis ct al., 1989: Sweasy et al., 1990) and also requires the heatshock regulated chapcronins G r o E I , and G r o E S (Donnelly and Walker, 1989; Liu and Tcssman, 1990). The biochemical roles of the U m u l ) ' and U m u C proteins in UV and chemical mutagenesis have not yet been established but genetic (Perry et al., 1985) and biochemical studies (Woodgatc ct al., 1990) have indicated that U m u C is able t o physically interact with U m u D and U m u l ) ' . On the basis of amino acid similarities of U m u l ) and U m u C to the bacteriophage T4 45, 44 and 62 gcnc products we have suggested (Battista et al., 1988, 1989) that the molecular action of these proteins may be related to those of DNA polymerase accessory proteins. Such a role lk)r U m u D ' and U m u C would fit well with the suggestion of Bridges and Woodgate (1984, 1985), which is based on physioh)gical and genetic studies, that the u m u D C gene products might act by allowing D N A polymcrase III holoenzyme to continue nascent chain elongation after a RecA-influenced misincorporation event opposite a lesion. An interaction of the u m u l ) C genc products with the I)NA-replication apparatus would bc consistent with our observations that lexA(Def) strains that carry a multicopy u m u D ' C" plasmid, and consequently ovcrexpress umuDC, exhibit a cold-sensitivity for growth that is associated with an inhibition of D N A replication after a shift to lower temperature (Marsh and Walker, 1985). We have recently reported that this cold-sensitivity can bc

suppressed by g r o H . and grol¢S mutations (l)onnelly and Walker. 1989). Genetic analyses of the . r e . D ( locus have been pivotal in attempts to understand the nmlect, lar basis of mutagenesis. The original t m m D and t#ntt(" point mutants were isolated by screening tot 4-nitroquinolinc-l-oxide (Kato and Shinoura, 1977), or UV (Steinborn. 1978) nonmutability. 'l'hcse mutations resulted in a loss of UV mutability and a decrease in resistance to UV. Two umu(" insertion mutations have been described. The tmm(.'121:Mu d l ( A p l a c ) n l u t a lion has been used in analysis of the regulation of the umul)(" operon (Bagg et al., 1981) and the umuCI22::Tn5 insertion mutation was used |tl generate a probe that facilitated the cloning of the umul)(' locus (Elledge and Walker, 1983a). Site-directed mutagenesis of tmml) was used to establish the physiological role of ReeA-mediated l.JmuD cleavage and tO identify key residues required for this phenomenon (Nohmi el al., 1988). Recently analyses of dominant umul) mutations further identified residues affceting U m u l ) cleavage and led to the suggestion that uncleavcd U m u I ) acts as an inhibitor of mutagenesis (Battista et at., 199(1). Genetic analyses of the mnuD(" mutants have been difficult because of the labor-intensive screens for nonmutability that have been used to idcntify many of the tmml) and umu(" mutations that have been analyzed (Kato and Shinoura. 1977, Stcinborn, 1978: Bagg el al., 1981: Battista c t a l . , 1990). In addition, many of the umul) and umu(" mutations that have bccn idenlificd in these screens have been recessive, loss-of-function alleles thal have becn of limited utility in genetic dissection of the roles of the t#nul)(" gene products in UV and chemical mutagcncsis. Wc and others have suggcstcd that the mntd)C gone products interact with a variety of other protcins so that mutations affccting subsets of these interactions might prove particularly i n f o f mative. In this paper, we describc a positivc scIcction for the isolation of m n u l ) ( mutations and a new screening proccdurc that allowcd us to isolate a number of ,mmD¢" allclcs with novel phenotypes. The properties of one of these. urmt('125, are described in detail. In addition, wc describe new phcnotypes associated with thc

185

prcscnce of multicopy urnuDC plasmids in E. coli strains. Materials and methods

Bacterial strains and plasmids Bacterial strains and plasmids used in this work are described in Table 1. Transductions using Plrir and other standard genetic techniques wcrc as described by Miller (1972). UV mutagenesis and survival was determined using supplemented M9 media as previously described (Bagg et al., 19811. Media and reagents Bacteria wcre routinely grown on LB medium at 37°C (Miller, 1972). Supplemented M9/glucose minimal medium with trace argininc was used for UV mutagenesis and survival measurements (Bagg et al., 1981; Miller, 1972). Kanamycin (Km) (25/zg/ml) and ampicillin (Ap) (100/zg/ml) were used to select for plasmid-containing strains (Marsh and Walker, 1985) and X-gal (5-bromo4-chloro-3-indolyl-/3-o-galactoside) was used to screen for /3-galactosidasc activity (Miller, 1972; Kenyon and Walker, 1980).

Hydroxylamine rnutagenesis In vitro mutagenesis of plasmid DNA was carried out in a reaction mixture consisting of 35 mg hydroxylamine hydrochloride, 9 mg potassium hydroxide and 1 /zg DNA in 0.5 ml H20 (Davis et al., 1980). This solution was incubated for 20 h at 37 ° C. DNA was precipitated by addition of 2 vol. ethanol on ice, and collected by centrifugation. The strain ABl157 was transformed with the mutagenized DNA by a modification of the CaCI 2 method (Mandel and Higa, 1970). The extent of mutagenesis was monitored using a plasmid, pLM1, carrying lacZ ÷ cloned into pBR322. Undcr the conditions described above, mutagenesis of pLM1 followed by transformation of a tester strain and subsequent plating on medium containing X-gal yielded about 5% white L a c Z - colonics. Many colonies that were darker blue or paler than normal were also observed, consistent with thc induction of random mutations in the cloned gene. We estimated the mutagenesis frequency under these conditions as roughly 10-4/nucleotide, though hydroxylaminc specifically induces G ' C ~ A : T transitions so only C : G base pairs are susceptible to mutagenesis (Davis et al., 1980).

TABLE 1 E. coli BACTERIAL STRAINS AND PLASMIDS

Strains

Relevant genotype

Reference or derivation

AB1157 GW21(KI GW 1103 GW110 KM 1190 GW5246 GW321~I GW3198 HBl01

argE3 umuDC ÷ AB1157 but urnuC122::Tn5 umuC::Mu d l (Ap lac) Mac ucrA GW1103 but ucrA '

Elledge and Walker, 1983a Elledge and Walker, 1983a By PI transduction Bagget al., 1981 Bagg et al., 1981 Elledge and Walker, 1983b KM1190x Ph'ir.GW2100 This paper By PI transduction By PI transduction l.aboratory strain

lexA5 l(Def) lex..451(Def) umuC122::Tn5 AB1157 but umuD44 AB1157 but urnuC36 recA(Def)

Plasmids

pSE117 pLM203 pLM206 pl.M207 pI.Ml

umuDC ', pBR322 ori. Km R, Ap R pSE117 but A u m u D C pSEll7 but A u m u C

pSE117 but Kn'l a region deleted lacZ + pBR322 derived, Ap a

Other plasmids described in text and Table 2.

Marsh and Marsh and Marsh and Marsh and This paper

Walker, Walker, Walker, Walker,

1985 1985 1985 1985

186

Selection ]'or mutant plasmids that fail to confer cold sensitit'ity Independent mutant umuDC plasmids unable to confer cold sensitivity on a lexA51(Def) strain (Marsh and Walker, 1985) were isolated by the following procedure: Independent cultures of KMI190(pLM2(17) were started from single isolated colonies growing at 43.5 ° C. These culturcs were incubated overnight at 43.5°C, diluted, plated and then incubated at 30 ° C. Plasmid DNA was prepared from pooled cells from plates having I(X)-I000 revertant colonies, and this DNA was used to transform fresh KM1190 cells. The resulting transformants were selected for ampicillin resistance at 43.5 ° C and then purified and screened for cold sensitivity. Approximately 50% of these transformants contained plasmids that failed to confer cold scnsitivity. One of these from each individual initial culture was chosen for further study. Dominant mutant plasmid screen DNA of the umuD+C ~ plasmids p S E l l 7 or pLM207 was mutagenized in vitro; the mutagenized plasmid DNA was then split into pools and transformed into strain ABlI57. A fraction of each transformation mix was plated onto selective media and incubated at 37 ° C until colonies wcrc well grown. Plates with 70-200 colonies were replica-plated onto minimal media plates containing trace (5/,tM) arginine and full amounts of the other nutrients required for AB1157 growth, and then UV-irradiated with 30 Jm -2. After incubation at 37 °C or 30 o C, replicated colonies were examined for Arg + papillae. The average replicated colony produced about 7 revertants in this screen. Those with significantly fewer or more Arg + papilli were rechecked with semiquantitarive patch and plate tests. Plasmid manipulations Plasmid DNA was purified by cesium chloride gradients or prepared by a rapid method (Maniatis ct al., 1982; Elledgc and Walker, 1983a). Plasmids for 'mix and match' mapping of the umuC125 mutation in mutant plasmid pLM109 (Fig. 5) were made by digesting DNA of the wild-type or mutant plasmids, partially or to completion, with appropriate restriction enzymes, gel

purifying the desired fragments, ligating, and transforming (Maniatis et al., 1982). The structure of the resulting plasmids was confirmed by restriction mapping. The plasmid pLM302 had the structure of pSE117 (Marsh and Walker, 1985) but a H i n d l l l - H i n d l I I fragment including part of the umuC gene originated from pLM 109 (Fig. 5). Likewise the plasmids pLM301 and pLM304 had the structure of pLM207 (Marsh and Walker, 1985) but a N c o l - H i n d l l l and a ClaI-HindlII fragment respectively derived from pLM 109 (Fig. 5). Several isolates of each construction were tested for the ability to confer UV sensitivity. The 615-bp fragment was sequenced as previously described (Battista et al., 1990). Maxicell preparations for identification of plasmid-encoded proteins and SDS-polyacrylamide gel electrophoresis were as described (Elledge and Walker, 1983a). Maxicell preparations were labeled with [3~S]methioninc for 15 min and promptly prepared for electrophoresis. SOS induction SOS induction was monitored by following /3galactosidase activity expressed from an SOS-inducible umuC121::Mu dl(Ap lac) fusion in strain GW1104 (Kenyon and Walker, 198(I; B a g g e t al.. 1981). Cultures growing at 3 0 ° C with an A~,~M~ of about 0.1 were exposed to 1 /.tg/ml mitomycin ('. Incubation at 3 0 ° C was continued and samples wcre withdrawn at various times and assayed for /3-galactosidase activity (Miller, 1972). Alternatively, cultures were treated with 1 fig mitomycin C / m l and allowed to grow for 2 h befl)re assaying. /3-galactosidase units were adjusted for cell density. Results

Mutant umuDC plasmids that fail to confer coldsensitirity for growth Our discovery that pBR322-derived plasmids carrying umuD+C ~ confer a cold-sensitivity for growth on lexA(Def) (Marsh and Walker, 1985) strains suggested to us that we might be able to use this phenomenon as the basis for a positive selection for umuDC mutants. This proved to be successful. We were able to isolate spontaneous mutant derivatives of such plasmids that failed to

187

neously arising plasmid mutants that fail to confer cold-sensitivity on a lexA51(Def) strain, pLM125 is defective in umuD function, pLM124 is defective in umuC function, and pLMI26 and pLM129 are defective in both umuD and umuC function. The plasmids pLMI23, pLM127 and pLM128 appear to be be partially defective in both umuD and umuC function. All of the u m u D C alleles we obtained in this selection were recessive with respect to their UV mutagenesis phenotype and in this respect differed from the dominant umuD alleles we have described recently (Battista et al., 1990). The fact that the plasmid pLM125, which carries a umuD mutation, and the plasmid pLM124 which carries a umuC mutation, do not confer cold sensitivity for growth on a lexA51(Def) strain (Marsh and Walker, 1985) suggests that high levels of both umuD and umuC are required for these phenotypes. Thus it appears this selection scheme will

confer cold-sensitivity for growth by (i) selection for derivatives that would not grow at 30 °C of a cold-sensitive lexA51(Def) strain carrying the pBR322-derived umuD +C + plasmid pLM207, (ii) plasmid isolation from pools of these derivatives, and (iii) retransformation of a lexA51(Def) strain. Independent isolates that failed to confer cold sensitivity were purified and retested. Agarose gel electrophoresis of each of the 7 mutant plasmids that we isolated suggested that they did not contain large deletions. These 7 plasmids were then transformed into AB 1157-derived strains carrying umuD44 umuC +, umuD + umuC36, or umuD ÷ umuC + alleles in their chromosomes and tested for UV mutability by argES ~ Arg- patch tests conceptually similar to those we have described previously (Perry and Walker, 1982). It is clear from Table 2 that we were able to obtain several different classes of mutant u m u D C plasmids by selecting for sponta-

TABLE 2 PHENOTYPES

OF MUTANT umuDC

P L A S M I D S IN a r g ÷ R E V E R S I O N

Relevant plasmid g e n o t y p e o f basis

Plasmid

UV-induced Arg ÷ revertants ~ chromommal genotype

o f isolation

umuD

pLM203

AumuDC

pSEI17 pLM207

umuD*C

PATCH TESTS FOR UV-MUTABILITY

- C"

umuD44C

+ + +

~

umuD

+ + +

-

-

+ + +

+ + +

+ + +

umuD*C*

+ + +

+ + +

+ + +

pLM123 b

cold-resistance

+ + +

+ +

+ + +

pLMl24 h pLMI25 h

cold-resistance cold-resistance

+ + + + + +

+ + -

+ +

pLMI26 pLMI27 pLM128 pLMI29

b b b b

cold-resistance cold-resistance cold-resistance cold-resistance

+ + + +

+ + +/-

+ + -

pLM101 pLMI08 pLM109 pLMll0 pLM112 pLM114 pLMl20

c c c c b " "

screen screen screen screen screen screen screen

+

in in in in in in in

+ + + +

+ C36

_

+ + + +

umuD+

C '

+ +

+ + +

+ + +

umuD+

C +

+ + + +

+ + + +

+ + + +

umuD*

C +

+ / -

+ / -

+ / -

+ +

+ + +

+ + +

+

+

+

+ +

+

+

+ + + +

+ + + +

+ + + +

umuD*C umuDumuD umuD*

~ C~ C" C +

I n d i v i d u a l t r a n s f o r m a n t colonies w e r e t r a n s f e r r e d by t o o t h p i c k to p a t c h e s o n m i n i m a l / g l u c o s e p l a t e s c o n t a i n i n g 5 ~,M a r g i n i n e a n d t h e n U V - i r r a d i a t e d . A t least 3 colonies of e a c h strain w e r e tested. b Derived from pLM207. c D e r i v e d f r o m pSE117.

188 provide an alternate means of isolating new mutant alleles of both u m u D and umtt(" by positive selection rather than by screening. A screen f o r u m u D C mutations causing h o t e l phetlotypes

Since the properties of the newly characterized u m u D C mutations identified in the above selec-

tion appeared similar to those of previously reported u m u D C mutations, we also developed a screen which we hoped would allow us to identify new alleles of u m u D C that exhibited different properties. Specifically, we searched for mutations in pBR322-derived plasmids carrying u m u O + ( "- that would change the apparent yield of UV-induced mutants of a strain carrying a single copy of u m u D + C - on its chromosome. We had observed that the number of Arg " revertants obtained after UV-irradiation of a u m u D ' C strain that also carried l t m u D * ( ' - on a multicopy plasmid, relative to thc number of Arg" rcvcrtants obtained after UV irradiation of a umuD'Cstrain without the plasmid, was quite dependent on conditions. In particular, the relative number of Arg" revertants obtained was a function of UV dose, temperature, and whether the bacteria being tested were grown on solid or liquid media. Empirically, wc found that similar numbers of Arg + revertants were obtained after treatment of either the u m u D " C ' strain AB1157 or strain AB1157 containing the pBR322-derived u m u D + C ~ plasmid, pSE117, with a UV dosc of 311 Jm 2 when the strains were grown on solid medium at 30 °C. We chose these conditions for our screen, reasoning that the number of A r g rcvertants of strains carrying a plasmid with a null mutation in u m u D C would be similar to that of a strain carrying a wild-type plasmid. Thus wc cxpccted that recessive, defective mutations would not bc detected in our screen whereas dominant mutations or mutations causing novel phenotypes would be. A total of about 70(10 colonies of the umul)'(" strain AB1157 carrying hydroxylamine-mutagenized derivatives of u m u D ' - C + plasmids were patchcd on minimal medium containing a trace of argininc, UV-irradiated with a dose of 31) J m - 2 and screened for Arg ~ revertants. 7 independent mutant plasmids were iso-

lated (Table 2): two (pLMI09 and p I . M l l 2 ) that greatly reduced the number of Arg" revertants obtained under the conditions of our screen, three (pLM101, p L M I I 0 and pLMI14) that moderately reduced the number of Arg" revertants obtained, and two (pI.M108 and pLM120) that increased the number of Arg + revertants obtained. We then examined the effects of these 7 plasmids on the mutability of u m u D 4 4 u m u ( ' - and u m u D " umu('36 strains as we had done abovc for the mutant plasmids obtained in the selection for cold resistance. A variety of different patterns of c o m p l e m e n t a t i o n / d o m i n a n c c arc evident in Table 2. Many, if not all, of the plasmids derived from this screen appear to not be simply defective for u m t t D C function but rather to carry u m u D C alleles conferring more complex phenotypes. p L M I 0 9 confers" increased sensitil'ity to killing hy U V on a lexA " strain

Because of its unusual phenotypc, wc decided to characterize more fully the behavior of one of the mutant plasmids, pLM109, which greatly reduced the number of Arg" revertants obtained under the conditions of our screen. This reduction in the number of Arg" revertants could be caused by an increased sensitivity of the strain to killing by UV-irradiation, by a failure of the processing of damaged DNA that gives rise to mutations, or by some combination of these. Because we had previously observed that overexpression of U m u l ) C in /exA(Def) strains caused lethality at low but not high temperatures (Marsh and Walker, It)85), wc therefore examined UV survival of our plasmid-containing lenA51 ' strains at both 3 0 ° C and 43.5°C. As shown in Fig. I, the plasmid pl,Ml09 greatly sensitized strains containing it to the killing effects of UV-irradiation. At 3 0 " C , a u m u C 1 2 2 : : T n 5 strain carrying pLMI09 was more UV-sensitivc than a strain carrying either the parental u m u D ' ( ' " plasmid pSEI17 or a strain carrying a plasmid with the u m u D C genes deleted, pLM203. We obtained similar results with the plasmids in a strain wildtype for the chromosomal umul)(" h~cus (data not shown). The fact that at 30 ° C the u m u C 1 2 2 : : T n 5 strain bearing pLMI09 was even more UV-scnsitive than the u m u ( ' 1 2 2 : : T n 5 strain

189 10

0!

8 u.

001 "

oool

1'o

~o UV

a'o

4'0

I0

Jm - 2

20 UV

30

40

Jrn - 2

Fig. 1. UV survival at 3 0 ° C (A) and 43.5 °C (B) of umuC122::Tn5 cells containing wild-type and mutant umuDC plasmids. Cultures were diluted in saline, spread on M9 minimal plates with trace arginine, UV-irradiated, and incubated at the indicated temperature. A, GW2100(pLM203); II, GW2100(pSE117); e, GW2100(pLM]09).

with a control plasmid lacking u m u D C genes demonstrated that pLM109 does not exert its effects solely by interfering with u m u D ÷ C +mediated repair. At 43°C, the survival of the umuC122::Tn5 strain after UV-irradiation was similar with or without pLM109, while p S E l l 7 increased resistance to killing by UV. Thus the strain containing pLM109 was more UV-sensitive than the strain containing p S E l l 7 at both 3 0 ° C and 43.5°C, but was most UV-sensitive at low temperature. It is interesting that, while the strains containing the u m u D ÷ C ÷ plasmid p S E l l 7 or the mutant plasmid pLM109 were both relatively more UV-sensitive at 3 0 ° C than at 43°C, the UVsensitivity of the parental umuC122::Tn5 strain carrying a A u m u D C control plasmid was relatively unaffected by temperature (Fig. 1). Thus, apparently a temperature-dependent component of sensitivity to UV killing is a characteristic of strains carrying the u m u D C genes cloned onto a multicopy plasmid.

p L M I 0 9 complements u m u C mutants for UV mutagenesis The plasmid pLMI09 complemented both umuD44 and umuC36 strains for reversion of argE3 ---, Arg ÷ at 5 Jm -z (Table 3). Fig. 2 extends these results and shows that at both 3 0 ° C and

TABLE 3 MUTAGENESIS AND SURVIVAL OF STRAINS EXPOSED TO 5 j m - 2 UV at 30°C Strains

Survival

UV-induced Arg ÷ revertants/ Survivor)< 10 6 a

AB1157 AB1157(pSE117) AB 1157(pLM 109) AB1157 umuD44(pLM109) AB1157 umuC36(pLM109) AB 1157 urauD44 AB1157 umuC36

0.96 0.94 0.46 0.54 0.51

5.6 13.6 9.4 5.8 7.8

1.0

O. 1

0.8

0.1

a Spontaneous reversion subtracted.

190 tO I 0

5.0.

50-

A

X 4.0"

4.0-

3.0-

3.0-

2.0-

2.0-

1.0

1.0"

o")

+ -1-

O~

10

20 UV

Jm

3'0 -2

4'0

0

0

2'0

~b UV

3'o

go

Jrn -2

Fig. 2. U V - m u t a g c n c s i s at 43.5 ° (" (A) and 311 ~ ( ' (B) in cells c o n t a i n i n g wild-type and m u t a n t umuD(" plasmids. C u l t u r e s were t r c a t c d as in Fig. I. A. GW210(Xpt,M2(I3k II, GW210(XpSE1171; o, GW21I)(XpLM109).

43 ° C pLM109 complemented umuC122::Tn5 for mutagenesis in a fashion similar to that of the parental u m u D + C + plasmid, p S E I I 7 . Thus, it appears that the low number of UV-induced Arg " revcrtants of pLMI09, containing strains we observed under the conditions used in our screen was caused by increased sensitivity of the pLM109-containing strains to killing by UV rather than by their failure to process damaged D N A in a way that gives rise to mutations. p L M I 0 9 fails to confer cold-sensitit'ity for growth As noted above, the plasmid pLM109 greatly sensitized strains to UV at 30 ° (" but not 43.5 o C. Sincc, as discussed above, we had previously found that the prescncc of the parental umuD ' C - plasmid in a /exA(Def) strain rcsuitcd in cold-sensitivity for growth (Marsh and Walker. 1985), wc wondered whcther the plasmid p L M I 0 9 would cause a similar cold-sensitivity for growth. Therefore, we introduced pLM109 into a lexA51(Def) strain. The lexA51(I)cf) strain was transformed at a typical frequency by pLM109 at 43 ° C. In contrast to the parent plasmid, pSE117, which lowered the viability of a lexA51(Def) strain at 3 0 ° C to 10-3-10 4 of the viability at 43°C, p L M I 0 9 did not confer cold sensitivity [60% viability of KM1190(pLMI09) at 30 ° C] although the

lexA51(Def) strain carrying pLM109 grew slowly at 3 0 ° C . Thus the mutation carried on pLMI(I9 apparently reduces the ability of the plasmid to cause cold-sensitivity for growth. Inhibition of induction o f the SOS response by multicopy u m u D C plasmids Wc have previously shown that the presence of m u c A B genes on a pBR322-dcrivcd plasmid intcrfcrcs with induction of the SOS response (Marsh and Walker, 19871. We therefore tested whether a similar phenomcnon was observed in cells carrying a pBR322-derived umuD ~C" plasmid. We obscrved that thc presence of such a u m u D ~ C + plasmid similarly inhibited induction by UV-irradiation, mitomycin (', and nalidixic acid of a variety of lacZ fusions to D N A damage-inducible genes (Kenyon and Walker, 198111 including one undcr the control of A cl repressor rather than LcxA (Table 4, Fig. 3; data not shown). The basis of this inhibition is not yet understood but it has not been observed with plasmids encoding only the umuD gene (Battista ct al., 1990) and thus does not appcar to be due to U m u D protein competing with LexA repressor for activated RecA protein. To test the effects of the fourteen umuD(" mutant plasmids described in this p a p e r on the

191 TABLE 4 EFFECT OF umuDC PLASMIDS ON SOS INDUCTION BY MITOMYCIN C IN A STRAIN CARRYING THE urauCl21::Mu dl(Ap, lac) FUSION Plasmid

/3-Galactosidase units/A~x ~ after mitomycin C induction "

pLM203 pSE117 pLM207

25 1.7 1.7

pLMI23 pLMI24 pLMI25 pLMI26 pLMI27 pLMI28 pLMI29

11 12 13 17 14 19 21

pLMI01 pLMI08 pLM109 pLM110 pLMII2 pLMII4 pLM 12(1

15 17 1.6 14 15 18 15

" Derivatives of GW1104 carrying the various plasmids were treated with mitomycin C (1 t~g/ml) and a~sayed for /3galactosidase as described in Materials and Methods.

induction of the SOS response we introduced each of them into the strain G W l l 0 4 which contains the umuC121::Mu dl(Ap, lac) fusion and determined /3-galactosidase levels after exposure to mitomycin C. Table 4 shows that only the plasmid pLM109 retained the ability to inhibit SOS induction strongly as measured by this limited test. More extensive characterization showed that both the umuD+C ÷ plasmid pSEI17 and pLM109 inhibited the induction of /3-galactosidase activity in umu121::Mu dl(Ap, lac) strains after exposure to UV-irradiation (Fig. 3A) or after treatment with mitomycin C at 3 0 ° C (Fig. 3B). A plasmid (pLM205) with umuD intact but umuC partially deleted did not inhibit SOS induction. The absorbance of each of the cultures continued to rise at similar rates during the course of the experiment so the failure of the umuDC plasmid-containing strains to accumulate /3galactosidase was not due to a general loss of protein synthesis capacity. Both umuD +C + plas-

mid-containing strains and pLM109-containing strains also exhibited reduced SOS induction by a plate assay for induction of /3-galactosidase by mitomycin C, though in this assay some SOS induction was observed. The plasmid pLM109 conferred moderate sensitivity to killing by mitomycin C in this plate assay. p L M I 0 9 affects sensitivity of a lexA(Def) strain to killing by UV-like the parental umuD +C + plasmid Since pLM109 was capable of inhibiting SOS induction in a lexA + strain, we wondered what the effect of pLM109 would be in a lexA(Def) strain, in which SOS functions are constitutively expressed. As shown in Fig. 4, the plasmid pLM109 conferred a slight UV-sensitivity to killing by UV-radiation to a lexA51(DeD strain at 37 o C strain but considerably less than in a lexA + strain. At 37 °C the lexA51(Def) strain carrying pLM109 was more UV-resistant than a lexA51 umuC122::Tn5 strain. At 37°C, the plasmid pLM109 confers a phenotype on lexA + strains that is intermediate between that observed at 30 o C and 43 ° C (data not shown). Even at 43.5 o C a lexA + strain carrying pLM109 is about as UVsensitive as a umuC36 strain (see Fig. 1). Unfortunately, the experiments could not be carried out at 30 o C, a temperature at which the phenotype of pLM109 strains was more pronounced, because of the poor growth of the lexA51(Def) (pLM109) strain at that temperature. The parental umuD+C + plasmid also conferred a slight UV-sensitivity on a lexA51(Def) strain (at 43°C) (data not shown) whereas it protected a lexA + strain. Thus, in contrast to the situation in a lexA +-defective strain, in a lexA(DeD strain the effects of pLM109 and a umuD+C + plasmid appear to be approximately similar. The mutation in p L M I 0 9 lies in umuC Since an increased sensitization to killing by UV has been observed under certain circumstances when mucAB or umuDC are present at high gene dosages or when the gene products are overexpressed (Elledge et al., 1983; Perry and Walker, 1982; McNally et al., 1990) we considered the possibility that pLM109 might carry a mutation that raised the copy number of the plasmid (Twigg and Sherratt, 1980), strengthened

192

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i

B 40.-

i A 0 0

40-

30; •

E

>

.= 300 ~_ ~2

20-

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o

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t6o

Time Post Irradlahon

t.Go

o .

.

0

.

.

80

(ms)

.

160

240

Time (ms)

Fig. 3. Induction of/3-galactosidasc activity at 30" (" in umu('12l::Mu dl ( A p / u c ) strains containing wild-type and mutant u,,nzd)( plasmids after treatment with A) I Jm 2 UV-radiation or B) mitomycin (" (I ,ug/ml). The arrows indicate the time at which the cells were exposed to UV or mitomycin was added. (A) • GW1103(pi,M203), • GW1103(pSE117), • GW1103(pl.M]09); (B) . GWll04(pLM203) [Aumul)(']; I. GWll04(pl.M205) [umul)+(" J, U. GWllf)4(pSEI17) [umt+D'('" }; • . GWI104(pLM304)

[tmlt:('125 ].

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Plasm~d umuD

umuC °

>

cI

~o.1-

-I

- - - pLM I 0 9

S

1

Nc H3 t

pLM30 1

I t

S

--- pLM302

R

pLM304

S

Fig. 5. Representation o f the u m u D ( ' region of p l M l 0 9 and mapping o f the umu('125 mutation, Bars underneath represent the section of plM109 DNA present in recombinant

0.01-

0.001

UV sensitiwty

tb

2'o UV

3'0

4o

dm -2

Fig. 4. UV survival of lexA(def) strains containing wild-type and mutant umuDC plasmids. Conditions were as in Fig. 1 but with incubation at 3 7 o ( `. A. GW5246 ( K M I I 9 0 tmmC122::Tn5): • KM1 lq0; • KMI It~XpLMI0'O).

umuD(" plasmids used to map the mutation of pLMI09. In each case, flanking DNA was derived from wild-type t4tvtul)(" plasmids regenerating an intact umt41)(" opercm (see Materials and Methods for details). The ability of each plasmid to confer UV-sensitivily (about 10-fold lower survival after irradiation with 20 Jm ? UV at 30"(" in a h,xASl+ strain) is noted. The parental wild-type plasmid is UV-resistant in this assay, kb. kilobase. Restriction sites; (', ('lab Nc. Ncoh t13.

Hindlll.

193

the u m u D C promoter, weakened the LexA binding site (Perry et al., 1985; McNally et al. 1990), stabilized one of the UmuDC proteins, or altered the properties of one of the UmuDC proteins. Decreased resistance to UV has also been associated with mutations in a 2-kb region upstream of the m u c A B region in pKM101 (Langer et al., 1985). About 1 kb of chromosomal DNA 5' to the u m u D C operon is present on the plasmid (pSEll7) from which pLMI09 was derived. To address these possibilities, we wished to know where the mutation on the pLM109 plasmid lay relative to the u m u D C genes. Therefore, we mapped the mutation by constructing hybrid plasmids in vitro in which part of the u m u D C

.;i.f..

operon and flanking sequences originated from pLM109 and part from plasmids carrying u m u D +C ÷. Fig. 5 summarizes the activities of the hybrid plasmids. The region of pLM109 carrying the mutation responsible for the phenotypes discussed above was localized to a 615-bp NcolHindlll fragment within the coding region of the u m u D C operon and spanning the u m u D - u m u C boundary. This mapping ruled out several of the possible explanations discussed above. DNA sequencing of this 615-bp region revealed that it contained a single a G : C ~ A : T transition mutation, umuC125, that altered codon 39 of u m u C from GCC to GTC and thus changed Ala39 to Val39. This Ala39 is conserved in the UmuC homolog MucB (Perry et al., 1985). We prepared maxicell-labelled extracts of a strain carrying the parent plasmid, pSEll7, and the mutant pLMl09 (Fig. 6). As our laboratory (Elledge and Walker, 1983a) and others (Shanagawa et al., 1983) have observed previously, the UmuC protein is less well expressed than the urnuD protein. The u m u C ÷ and u m u C 1 2 5 gene products appeared identical under the labeling conditions used suggesting that the u m u C mutation alters the properties of the UmuC protein but does not have a major effect on its stability. Discussion

L

'!

.,~ UmuC

•4 t I~la

.,~ UmuD

Fig. 6. Autoradiogram of [35S]methionine-labeled maxicell extracts prepared from HB101 carrying either the u m u D ~ C ÷ plasmid pSE117 or the mutant pLMI09; proteins were separated by SDS-p.(flyacrylamide gel electrophoresis. Positions of wild-type UmuC and UmuD proteins as well as /3-1actamase (Bla) are indicated.

We have described two approaches for the isolation of new alleles of cloned E. coil u m u D and u m u C genes. The first approach is based on the cold-sensitivity for growth conferred by a multicopy u m u D + C ÷ plasmid in a /exA(Def) strain and represents the first positive selection for u m u D C mutations. Our preliminary results indicate that mutant plasmids isolated using this screen carry a variety of classes of u m u D C alleles. The second approach is based on screening a u m u D +C + strain carrying a multicopy u m u D ÷C ÷ plasmid for apparent alterations in UV-mutability. Although this screen was more labor intensive than the selection, it yielded a number of potentially interesting u m u D C mutations, some with properties different from previously isolated u m u D C mutations. The properties of one of these, umuC125, which is present on the plasmid pLM109, are described in this paper. Since

194

pLM109 does not confer cold sensitivity on a /exA(Def) strain, presumably plasmids carrying this class of u m u C mutation could also have been identified by the use of the first approach. These studies have focused our attention on the novel phenotypes exhibited by cells containing multicopy u m u D C plasmids and on the temperature dependence of certain of these phenotypes. As we have discussed, /ex.A(Def) strains carrying a multicopy u m u D - C ÷ plasmid exhibit a cold-sensitivity for growth that is associated with an inhibition of DNA replication upon a shift to lower temperature (Marsh and Walker, 1985). Our isolation of a derivative ( p L M I 2 4 ) o f the u m u D ÷ C ÷ plasmid pLM207 that carries a u m u C mutation and fails to confer cold-sensitivity for growth to a lex.A(Def) strain, supports our previous conclusion (Marsh and Walker, 1985) that the u m u C gene product is required for the cold-sensitivity for growth phenotype. Our isolation of a derivative (pLM125) of plasmid pLM207 that carries a u m u D mutation and fails to confer cold-sensitivity for growth raises the possibility that this phenomenon also requires the u m u D genc product. However, we have not yet ruled out the possibility that the u m u D mutation on pLM125 eliminates cold-sensitivity by a polar effect on u m u C expression. In this paper, we have also dcscribed novel phcnotypes of l e x A ' strains that carry multicopy u m u D ÷ C ÷ plasmids. We had previously reported that the presence of a multicopy m u c A ~B ~ plasmid in a lexA ÷ strain inhibited SOS induction in response to UV-irradiation and other SOS-inducing agents (Marsh and Walker, 1987). We have now shown that the presence of a multicopy u m u D ÷ C ÷ plasmid in a lexA ÷ strain similarly inhibits SOS induction. The molecular basis of this inhibition of SOS inductkm is not yet understood but it is clear that it is not due to the u m u D protein alone outcompeting LexA for the pool of activated ReeA since a multicopy plasmid carrying u m u D but not u m u C does not inhibit SOS induction (Battista et al., 1990). At first, there might appear to be a contradiction between the observation that muiticopy u m u D ÷ C ÷ plasmids inhibit SOS induction in lexA + strains and the observation that such strains are UV-mutable since UV-mutagenesis requires RecA-mediated

LexA cleavage and RecA-mediated UmuD cleavage. However, it appears that the inhibition of SOS induction is not complete. Western blots of UV-irradiated lexA ~ cells carrying a multicopy u m u D ~ C ' plasmid, which were performed using antiserum raised against UmuD, have shown that the level of u m u D C expression in these cells is higher than in lexA + cells lacking the plasmid (J.R. Battista and G.C. Walker, unpublished results). Presumably this is a consequencc of the increased dosage of the u m u D + C - operon. Furthermore, these studies have shown that, although the rate of UmuD cleavage is reduced in these cells relative to cells that contain a multicopy plasmid carrying u m u D only (Battista et al., 1990), the level of U m u D ' in UV-irradiated cells carrying a multicopy UmuD ~C ÷ plasmid is higher than that in a similarly UV-irradiated lexA ÷ strain lacking such as plasmid. We and others have considered the possibility that the u m u D C gene products are involved in more than one process affecting mutagcncsis a n d / o r survival after DNA damage. We have concentrated on the characterization of one particular u m u ( " mutation, u m u C 1 2 5 , that is carried by the plasmid pLMI09 since its properties appear to bear on this issue. This mutation changes Ala39 to Va139. Based on analyses of UmuC125 expression in maxicells, it does not appear that this missense mutation has any effect on the level of expression or stability of the UmuC protein. Thus it seems likely that the properties of u m u C 1 2 5 mutants are duc to some qualitative alteration in the properties of UmuC or some UmuC-containing complex rather than duc to some quantitative change in the level of activity of UmuC or some UmuC-containing complcx. The u m u C 1 2 5 mutation is unusual in that plasmids carrying this mutation sensitize l e x A ' cells to killing by UV-radiation but are still ablc to complement the deficiencies of u m u C mutants in UV-mutagenesis. Furthermore, multicopy t t m u D C plasmids carrying the u r n u C 1 2 5 mutation cause very little cold-sensitivity for growth when present in/exA(Def) strains yet retain their ability to inhibit SOS induction when present in /exA(Dcf) strains. It is interesting to note that multicopy m u c A ~B ~ plasmids cause little coldsensitivity for growth when present in lex.A(Dcf)

195 strains but inhibit SOS induction when present in lexA ÷ strains. Thus, with respect to these latter two phenotypes, the urnuC125 mutation makes the properties of the umuD+C125 plasmid resemble those of a mucA +B ÷ plasmid. The observation that multicopy urnuDC plasmids carrying the umuC125 mutation sensitize ceils to killing by UV but retain their ability to complement the deficiencies of umuC mutants in UV-mutagenesis is intriguing. Our observations indicate that this sensitization of ceils to killing by UV is a lexA +-dependent phenomenon. In particular, we observed that a multicopy plasmid carrying umuD + umuC125 sensitizes lexA + strains to killing by UV whereas a umuD + u m u C - plasmid does not and that both plasmids have similar effects on sensitivity to killing by UV in/exA(Def) strains. This conclusion is consistent with models proposing that part of the UV-sensitivity conferred by the umuC125 mutation is due to inhibition of the SOS response or to inhibition of some SOS-inducible repair response that is present in excess in /exA(Def) strains. It seems inconsistent with models in which the u m u C l 2 5 gene product is toxic after UV-irradiation since there should be more of the umuC125 gene product in lexA(Def) strains than in lexA + strains. The possibility that the u m u D C gene products could influence cellular survival after UV-radiation in a fashion that is genetically separable from their role is UV-mutagenesis is consistent with certain previously reported observations. Witkin et al. (1987) have reported that, under certain conditions, the u m u D C gene products can influence the recovery from the inhibition of DNA replication that occurs after UV-irradiation. This phenomenon has been termed induced replisome reactivation (IRR) (Khidhar et al., 1985) and is SOS-inducible but does not normally require u m u D C function. However, in a recA718 mutant, IRR has been shown to be dependent on umuC function (Witkin et al., 1987). Thus it is possible that the umuC125 mutation alters UmuC in a way that permits it to perturb IRR in a recA + background. In a separate study, Liu and Tessman (1990) have reported that the SOS-inducible, umuDC-dependent reactivation of UV-irradiated single-stranded bacteriophage S13 that occurs in groEL and groES backgrounds is not mutagenic

whereas the reactivation occurring in groE+S + backgrounds is mutagenic. These observations suggest that the u m u D C gene products can participate in some class of DNA-repair process that does not involve the introduction of mutations. Thus it is possible that the umuC125 mutation differentially affects the ability of the UmuC protein to participate in an error-free process that affects survival after UV-damage without affecting its ability to participate in the type of processing in which mutations are introduced. With respect to this possibility, it is interesting that the groES and groEL are heat-shock genes whose expression is strongly influenced by temperature, that several of the phenomena discussed in this paper exhibit temperature-dependent effects, and that we have previously shown that groEL and groES mutations suppress the cold-sensitivity for growth of lexA(Def) strains carrying multicopy u m u D + C + plasmids (Donnelly and Walker, 1989). Also, we have previously shown that a multicopy umuD +C + plasmid complements umuD uz'rA and umuC ut'rA strains for UVmutagenesis but does not increase their resistance to killing by UV-radiation (Elledge and Walker, 1983a). Finally, we have recently shown that an operator mutation that reduces LexA binding upstream of the mucAB operon, and therefore leads to increased mucAB expression, increases the susceptibility of cells to mutagenesis but also increases the sensitivity of ceils to killing by UV (McNally et al., 1990).

Acknowledgements This work was supported by Public Health Service Grant CA21516 from the National Institutes of Health. L.M. was supported by Training Grant ES07020 from the National Institutes of Health. S.H. participated though the Undergraduate Research Opportunities Program at M.I.T.

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196 mutagencsis, in: E.C. Friedberg and P.('. Hanawalt (Eds.I. Mechanisms and Consequences of DNA Damage Processing, Liss, New York, pp. 255-459. Battista, J.R., T. Nohmi, C.E. Donnelly and G.('. Walker (1989) Amino acid similarities to other proteins offer insights into the roles of UmuD and UmuC in mutagencsis, Genomc. 31,594-596. Battista, J.R., T. Ohta, T. Nohmi, W. Sun and G.C. Walker (1990) Dominant negative u m u D mutations descreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 87, 7190-7104. Bridges, B.A., and R. Woodgate (1084) Mutagenic repair in Escherichia coll. X. The u m u C genc product may bc required for replication past pyrimidinc dimers but not for the coding error in I_IV mutagenesis, Mol. Gcn. Genet., 196, 364-366. Bridges, B.A., and R. Woodgate (1985) Mutagenic repair in Escherichia coil: products of the recA gene and of the u m u D and u m u C genes act at different steps in UV-induced mutagenesis, Proc. Natl. Acad. Sci. (U.S.A.), 82, 4193-4197. Burckhardt, S.E., R. Woodgate, R.II. Scheuermann and II. Echols (1988) UmuD mutagenesis protein of l-scherichia coli: overproduction, purification and cleavage by RecA, Proc. Natl. Acad. Sci. (U.S.A.). 85, 1811-1815. l)avis, R.W., D. Botstein and J.R. Roth (19811) Advanced Bacterial Genetics, Cold Spring |larbor Laboratory, Cold Spring llarbor, NY. Donnclly, C.E., and G.C. Walker (1989) grot', mutants of Escherichia coil are defective in u m u D C - d e p e n d e n t UV mutagenesis, J. Bacteriol., 171, 6117-6125. Dutreix, M., P.L, Moreau, A. Bailone, F. Galibert, J.R. Battista, G.C. Walker and R. Devoret (1989) New recA mutations that dissociate the various RecA protein activities in Escherichia coil provide evidence 1~3ran additional role for RecA protein in UV mulagenesis, J. Bacteriol., 171, 24152423. Elledge. S.J., and G.C. Walker (1983a) Proteins required for ultraviolet light and chemical mutagenesis: Identification of the products of the u m u C locus of Escheriehia coll. J. Mol. Biol. 164, 175 192. Elledge, S.J., and G.C. Walker (1983b) The muc genes of pKM101 are induced by DNA damage, J. Bacteriol., 155, 1306-1315. Ennis, D.G., N. Ossian and D.W. Mount (1989) Genetic separation of Escherichia coil recA functions for SOS mutagenesis and repressor cleavage, J. Bactcriol., 171, 2533-2541. Kato, T., and Y. Shinoura (1977) Isolation and characterization of mutants of £~cherichia coil deficient in induction of mutations by ultraviolet light, Mol. Gen. Genet. 156, 121-131. Kenyon, C.J., and G.C. Walker (1980) DNA-damaging agents stimulate gene expression at specific loci in Escherichia coil, Prec. Natl. Acad. Sci. (U.S.A.), 77, 2819-2823. Khidhir, M., S. Casaregola and I.B. Ilolland (1985) Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coil: inhibition is independcnt of rec,4

whilst recovery requires RecA protein itself and an additional, inducible Sf)S function. Mol. Gem (ienet., ITS9. 133 141). Langer. P.J., K.L Pcr~' and G.('. Walker (1985)('omplenlentation of a pKM101 derivative that decreases resislance to UV killing but increases susceptibility to mutagenesis. Mutation Res.. 1511, 147-158. Liu, S.-K., and I. lessman (199ll) I-rror-prone repair can be error-free, J. Mol. Biol., 216, 803-.8117. Mandel, M., and A. Higa (1970) Calcium-dependent bacteriophage DNA infection, J. Mol. Biol. 53, 150-162. Maniatis. "1'., E.F. Fritsch and J. Sambrt~k (1982) Molecular Cloning, a Lalx)ratory Manual. Cold Spring Ilarbor l.aborator3', ('old Spring llarbor, NY. Marsh, L., and G.('. Walker (1985)Cold sensitivity induced I'b overproduction of UmuD(" in l','scherichm coil, J. Bacte riol., 162. 155 161. Marsh, L.. and G.('. Walker (1987) New phenotypes associated with mucAB: alteration of MucA sequence homologous to the [,exA cleavage site, J. Baeteriol., 169, 1818 1823. McNally, K.P., N.E. Frcitag and (i.C. Walker (1990) LcxA-independent expression of a mutant mucAB operon. J. Bacteriol., 172, 6223-6231. Miller, J.H. (1972) Experiments in molecular genetics. ('old Spring Harbor Laboratory, Cold Spring I larhor, NY. Nohmi, -1., J.R. Battista, I..A. Dodson and G.C. Walker (1988) RecA-mediated cleavage activates umuD for mutagenesis: mechanistic relationship between transcriptional dereprcssion and posttranslational activation. Prec. Natl. Acad. Sci. (U.S.A.), 85. 1816-1821). Pcrry, K.t.., and G.('. Walker (1082) Identification of plasmid (pKMll)I)-coded proteins inw~lvcd in mutagcnesis and UV resistancc, Nature (London). 3IX}, 278 281. Perry., K.I,., S.J. Elledge, B.B. Mitchcll. I.. Marsh and G.('. Walker (1985) umuD(" and muc,,1B operons whose products arc required for UV light- anti chemical-induced mutagcnesis: UmuD, MucA and l,exA proteins share homology, Proc. Natl. Acad. Sci. (U.S.A.), 82, 4331 4335. Peterson, K.R.. N. ()ssanna, A.T. Thliveris. D.G. Ennis and D.W. Mount (1988) Depression of specific genes promotes I)NA repair and mutagenesis in Escherichia coli, J. Bacteriol.. 170, 1-4. Shinagawa, tl., T. Kato, T. lse, K. Makino and A. Nakata (1983) Cloning and characterization of the umu opcron responsible for inducible mutagenesis in t-scherichia coll. (iene, 23, 167-174. Shinagawa, H., FI. lwasaki, I'. Kato and A. Nakata (1988) RecA protein-dependent clcavage of UmuD and SOS mutagenesis, Prec. Natl. Acad. Sci. (U.S.A.), 85, 181~',181(). Stcinborn, G. 11978) Uvm mutants of Escherichia coh KI2 deficient in UV mutagenesis, I. Isolation of uum mutants and their phenotypical characterization in DNA repair and mutagenesis, Mol. Gen. Genet., 165, 87-93. Sweasy, J.B., E.M. Witkin, N. Sinha and V. Roegner-Manisealco (1990) RecA protein of I-scherichia coli has a third essential role in SOS mutator activity, J. Bacteriol., 172. 31)3(}-3()36.

197 Twigg, A.J., and D. Sherratt (1980) Trans-complementable copy-number mutants of plasmid ColEI, Nature (London), 283,216-218. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Walker, G.C. (1985) Inducible DNA Repair Systems, Annu. Rev. Biochem., 54, 425-457. Walker, G.C., and P.P. Dobson (1979) Mutagenesis and repair deficiencies of Escherichia coli umuC mutants are sup-

pressed by the plasmid pKMI01, Mol. Gen. Genet., 172, 17-24. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869907. Woodgate, R., M. Rajagopalan, C. Lu and H. Echols (1989) UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD', Proc. Natl. Acad. Sci. (U.S.A.), 86, 7301-7305.