Regulation of damage-inducible genes in Escherichia coli

J. Mol. Biol. (1982) 160, 445-457

Regulation of Damage-inducible Genes in Escherichiu coli

CYSTHIA J. KEXYOX’, ROGER BREXT’. MARK PTASHNE*

ASI) (:KAHAM c. WALKER’

‘Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, F.S.A.

e t o Biochemistry, Harvard Cbiversity InepartmP~mkdge, MA 02138, 1.9.d. : A

(Received 3 March 1982, and in revised form 2 June 1982)

The expression of the din.4, dinR. dint) and dinF gr~les of Escherichia coli is stimulated by certain agents that either damage DNA or block DNA replication. These agents induce the complex SOS response. Genetic evidence is presented to show that the expression of each of these genes is blocked in uninduced cells by the same repressor. the lezil protein. In addition, the regulatory regions of the din.4 and d&B genes were cloned, and the approximate positions of the promoters were determined. Purified lexrl protein was found to block transcription of these genes ill vitro.

1. Introduction

There are instances in many different biological syst’ems in which a particular environmental signal elicits a complex response that’ involves multiple physiological changes. One of the best-studied examples of such a co-ordinated cellular response occurs when the DKA of Escherichia coli is damaged or DNA replication is blocked by any of a variet’y of agents (for reviews, see Witkin, 1976: Gottasman, 1981). Under these conditions, several different DNA repair systems are induced as well as a system that is necessary for mutagenesis by ultraviolet irradiation and many types of chemicals. In addition. cell division is blocked, and t,he cells begin t’o grow filamentously, accumulating up to 16 chromosomes. Also, certain integrated bacteriophages are induced and begin to grow lytically. These and other inducible functions appear to be an adaptive response to a (dangerous) environmental condition. Because some responses at least appear to promote cell survival, they have been called collectively the SOS functions (Radman, 1975: Wit~kin. 1976).

The mechanism by which bacterial cells can regulate the expression of these metabolically unrelated processes has been studied intensively in a number of laboratories, and a fairly detailed picture has begun to emerge. The SOS functions are controlled co-ordinately by two regulatory elements. the recA and 1rxA

445 0022%2836/82/270445-13 $03.00/O (‘ I982 Xradrmic Press Inc. (London) Ltd.

446 (‘. J h;EN\.OS R7’ .I/,

proteins: mutations that, alter either protein ca,n prevent the expression of these responses. The ZPXA protein appears to function exclusively as a regulator of the SOS response. while the red protein also functions Tao promot,e homologous recombinat,ion. In response to SOS-inducing treatments. the expression of a number of genes is stimulated. These include the rw.4 (McEntee. 1977: Gudas & Mountj. 1977; Emmerson & West. 1977). lrxd (Brent & Ptashne. 1980: Littjlr & Harper. 1979). ~mrrC (Bagg et al.. 1981 ), UVK (Kenyon & CValker. 1981 : Fogliano R Schendel. 1981). ~$4 (Huisman $ D’Ari. 1981) and him4 (H. 1. Miller4 nl.. 1981) genes, In addition. we have ident,ified a number of damage-inducible (din) pews on the basis of t,heir inducibilit’y by the DNA-damaging agent mitomycin C‘ (Krnyon 8.z Walker. 1981). These include the dind . din,B. ninD. dinE’and 1rer.4 genes. Although the functions of some of these genes have not yet been determined. others are known to code for products that are necessary for the occurrence of partiwlar SOS processes. Thus. it has been possible to study the regula,tion of the physiologicall> complex SOS response by focusing on the regulation of the inducible genes.

The induction of each din gene ident’ified has been shown to involve the two SOS regulatory elements. the wed and IPX~-I proteins. The mwhanism I,- which these two proteins regulate the expression of three transcriptional unit,s (the wc=l. ir~,.4 and prophage operons) has been dedwed from a series of genct,ic and biochemical experiments. We have shown by genetic and biochemical techniques t,hah the wczI and Irx:d genes are repressed by the l~z.-i gene product (Brent & Ptashne, 1981: &o LittIle et aZ., 1981). Lysogenic bact,eriophages. such as prophage A. arr repressed by, phage-coded repressors. The initial event, that leads to the derepression of these genes appears to be the activation of the m-,-l protewe function. which ov~~urs in response to an unidentified signal arising from S( )S-inducing treatment~s. The rwA protease specifically csleaves prophage r’cywsors (Kobcrts f4 (xl.. 1978). as well as t,he ba&erial IPX,~ prot,ein (Little et ni.. 1981). Thus the way in which 1)NA damage leads t,o the rxpression of these gems is by sCrnulating thr wt.-l prot,clirl to inactivat’e their repressors.

In this paper we report a series of genetic and biochemical studies designed to determine more precisely the way in which the various din penes are controlled. LI’o present1 evidence indicating that each gene is represswl t)y the same regulator> molecule. the lex.4 protein.

2. Materials and Methods

(a) Ructrriol rend plusmid .sfrtr ius

Hactrrial strains and plasmids are listed in Table I. ‘The /lit, : : .2lud fusions 1% ere isolated ill strain UW1000. which carries a deletion of the lac%ow apron. a sfi.-l I/ mutation to rcbduc~l filamentation in the presence of SOS-inducing agents. and thr wc.-1441 (&f-l) mutation III /PXA + cells. thr rec.-l442 mutation causes expression ofthc SOS response at high temperaturc~ (42’ C). but not, at 3OY’. where all grnet,ic. manipulations were performrd.

Bacteriophage 1’1 transductions were essrntially as drwrihed by Xlillrr (1!)71). l’hts ap-Sl mutat,ion was introduced into derivatives of the fusion strains carrying wc=1441. mnlb’ : : l’rt:i and /ax.-l;j mutations (see Kenq’on & Walker, 1981). Xlal’ transductants were’ selrcted

DAMAGE-INDVCIBLE GENES

TABLE 1

Bacterial and plasmid strains

447

Strain Relevant markers Source or Reference

GW1000

c:w1010 c:w1011

GWlO12

(:wlOl3

GW1014

GW1030 GW1040 GW1070 DB6659 DM1187 (:wlOOl GW1002 (:w1003 GW1004

lacd(lJlSR), reci1441 (Cf.1), .$A11 GWlOOO. but dinA : : Mud GWlOOO. but dinA : : Mud, recA56, srl : : TnlO GWlOOO, but d&Al : : illuti lerA3. n&E : : Tn5 GWlOOO. but d&Al : : Mud spr-51 GWlOOO, but d&Al: : Mud spr-51. recA.56. srl : : TnlO GWlC@O, but d&B1 : : Mud GWlOOO. but d&l)/ : : Mud GWlOOO, but d&F1 :. Mud recA56. srl : : TnlO Spr-51 GWlOOO/pGW51O(dinA-Inc) GW1000/pGW511 (I’&,<.,) GWlOOOJpGW520 (d&B-lnc) UWlOOO/pGW521 (I’,,,,,,!)

Krnyon & Walker (1981)

Kenyon & Walker (1981) Text

Kenyon 8r Walker (1981)

Text

Text

Kenyon & Walker (1981) Kenyon & Walker (1981) Kenyon & Walker (1981) 1). Hotstein D. Mount Text Text Ttlxt Text

Strains were constructed as described in Materials and Methods. or in the legend to Fig. 2. Derivatives of the fusion strains carrying recA56 : IezA3: spr-52 : and spr-51 and raci156 mutations were obtained (and arc numbered) as indicated for thP dind fusion strain drrivatirrs.

following infection with phage Pl grown on the spr-51 mutant, DM1187. The strains were tested for the u.v.t-resistant Spr phenotype (the lezil3 parent strains are u.v.-sensitive). In t,he recA441 background, the STS (spr) phenotype could be verified by spotting wild-type X onto a lawn of the candidate strain. Phage spotted onto a lawn of cells carrying a Zez.43 mutation give rise to a turbid spot while phage spotted onto cells carrying spr- mutations generate clear disks in the bacterial lawn. The recA56 mutation was introduced into the fusion strains by selecting for tetracycline-resistant transductants following infection with 1’1 grown on strain DB66S (srl : : T&O. rrc1456). RecA - derivatives were u.v-sensitive.

(c) Assay for /+-galactosidase

The assay procedure used was adapted from that described by Miller (1972). The /Z- galactosidase activity in a culture was determined by withdrawing a 1 ml sample and adding 0.5 ml to 05 ml assay buffer containing 100 pg chloramphenicol/ml (on ice) and the remaining 0.5 ml to 0.5 ml of a solution containing a 1 : 200 dilution of formaldehyde in water. Cells were assayed within 3 h after their removal from the culture. Sodium dodecyl sulfate (40 ,.~l,Ol~/~) and 40 ~1 of CHCl, were added to the assay tubes, and the solution was vortexed for 10 s and then kept at room temperature for 10 min. The solution was warmed to 29°C and 40 ~1 of a 4 mg/ml solution of o-nitrophenyl-j%n-galactoside was added to start the reaction. Reactions were terminated by adding 0.5 ml of 1.0 M-Na,CO,. Cell debris was removed by centrifugation, and the absorbance of the solution at 420 nm was determined.

t Abbreviations used: u.v.. ultraviolet irradiation: kb. lo3 bases or basr-pairs: b.p., base-pairs

448 (' .I. KblN\r.ON /i't' .I/

The wll density ~vas detrrminrd hy tnvasuring t,hv absort~att~c~ of t,ttti formald~~tt~dc~~tt~r~~~t~~tl ct~lls at 600 tltt,.

(341s (200 ml) wre grown owrnight in LIS medium. (‘PIIS ww collwt~d t)y wntrifugatiott and wsuspendrd in 7.5 ml of a solutiott cwtttainittg 50 mw’l‘ris (pH 8). IO0 ~~~l31)T.A. at~d dO”;, sucrose. Lysozytnt) (10 pg) in 1.8 ml of 025 wTris (pH X) was added and thv mixture ~vas incubated for 15 min ott iw. RNAase A (IO ~1 of a 21 ~g/ml solution). proteinaw K (2 trig in 1 ml of 0% al-Tris. pH 8) and sodium Sarkosyl (IO ml of a I”,, solution containing 7.5 tttw EDTA) ww added. and the suspension was inwttat~t~d at FEY‘ for 6 h. (“~(‘1 BYSS addrd to a density of 1.7 g/‘cm3. Thr samples w~rv wnt,rifuged at 12.000 rwsitttin for I6 h in a \Y’i;iO rotor. I>NA was c~ollrctrd hi puncturittg the bottom of’ t,hts tultt. with a hypodermic. ttcwllv attd cwllect,ing the highly vtscous portion of thr gradient. The UN.4 ~.as dialyzed agaitrst three l-l vols of IINA buffer (10 mwTris. 3 trt.\t~Xa(‘l. I tnwF:I)TA. pH 8). This pwparat,iotr yielded about I mg of high molecular wipht l)NA. Occasicmally thv I)NAA was partialI> resistant to digestion with r&ric%ion vnzymw. 1 tt this ww. samples (itt Eppendorf t,tthw) were tlxtracztrd wit,h phenol. c~xt~rac*tc~ti \\.ith ~hlorofortn attd thrtt precipitated with t>thatlol.

(e) I’rrptrrtrtiofc of’ plnsm id //.\‘.A

Plasrnid I)K’A ~vas pwpared using a slight ttroditicat iott of thus procrduw of (‘ltw till & Helinski (1970).

(f’) (‘lo,, it/q

In the initial cloning of thtl nip : : Irrc fusions. pHK:%I:! attd c~hrotnosotnal 1)S.A \\~~t’,s digested with RnmHl. Samples were precipitated with cthattol and 20 pg chrotnosomal iJNi\ and 5 pg plasmid DNA were resuspended in IO0 ~1 ligat,ion buffer (.50 mar-Tris. HCI (pH 7%). 10 mwMgC1,. 20 mM-dithiathreit,ol. I ,O tnwAT1’. and 60 pg horiw serum all~umitt;‘ttil). Ha&riophage T4 IlKA ligaw \vas added. and thr wactions UYI’~’ incubated at 16°C for 12 h. Competent (~WIOOO cells awe transformed with the ligation mixtuw. and transformants wena selected on plates containing ampicillin (20 pg,/ml) attd -1-O pg ;i-ltrotno--C-c~hl~)ro~:~~ indo)-I-/St,-galactosidp (X-gal)/ml (Ha&m).

(g) Isolutiotl oj rrstrictiotc jrtrgtrteut.u

Isolation of rrstrict,ion fragments aftrr rl(,c:troF’horesis ott -CO,, 1 )olyacr\-latnidc grls atrtl I staining with mrthylenr blw (Maniatis rt t/l.. 1975) was as d~wrihc~d by Maxam & (:illwt (1980) with minor modificat,ions.

(h) In vitro tmuscriptior~s

Repressiott of t,ranscript~ion of wstric+ion fragmtants I)y I~x.4 protein it/ cv’tro was essentially as described (Brent & l%ashne. 1981 ) except that the reaction mixtures contained RNA polymerasr (a gift from W. McClutv) at 50~~. CTI’ at 5 PM. nnlabelcd HTTP at 2.5 +I. [ I-~‘P/UTP at 2.5 PM, and sometimes conta,inrd hoviw serum albutnin at 50 +I. Aftrlt rlrctrophoresis on 8 >vurra/P o polyac~rylamidc gels (Maxam B (:ilhert. 1980) tranwripts were visualized by autoradiography on XAR-5 film. /<,X.-I protein was purified as drsc~rihrd by Brent & I’tashne (1981).

449

83. Results

(a) Genetic studies

LVe originally identified the d&4, d&R, d&D and d&F genes by a method t)hat allows one to probe the genome for transcriptional units that are expressed ill response to a given environmental stimulus (Kenyon 8: Walker, 1981). Random cellular promoters were fused to the lac structural genes using the Mud (Ap, Zac) operon fusion vector (Casadaban & Cohen, 1979). Cells that carried fusions of Znc to genes induced by DNA-damaging agents (din genes) were identified as colonies that synthesized fl-galactosidase in response to the SOS-inducing agent mitomycin (‘. Although the functions of these genes are not known, their regulation can be st,udied by measuring the level of /3-galactosidase produced under various conditions. Because the Mud (Ap, lac) operon fusion vector generates operon fusions, in which the wild-type /3-galactosidase protein is translated from a hybrid transcript, the levels of /3-galactosidase in the fusion strains reflect the relative rates of transcription in the fusion strains. In this section, we describe a genetic: experiment that analyzes the way in which the two SOS-regulatory elements. thr rrcrl and 1exA proteins. regulate din gene expression.

The SOS response can be blocked by &l-l3 mutations, which alter the lrzil protein in such a way that it cannot be cleaved by the recA protease as efficient’ly as wild-type 1exA protein (Little et al.. 1981). This mutation blocks the induction of dir? gene expression (Kenyon & Walker, 1981). Because the 1exiZ protein is known to function as a repressor. it is reasonable to assume that t’he ZexA3 mutation blocks induction because the lexA protein represses a gene or genes necessary for din gene expression. There are at least t,wo ways in which t’he ZexA3 mutation protein might) block din gene expression in uninduced cells. The brxd protein could dire&l) repress individual din genes. Alternat,ively, other cellular repressors might bind t’o the din genes. In this case, lexA3 mutants might be uninducible because the altered lexil protein blocks the synthesis of sufficient amounts of recA protease to remove

the ac,tual din repressors. This appears to be the mechanism by which the le&3 mutation reduces the induction of prophage gene expression (Casbellazzi rt al.. 1972).

A genetic test can distinguish between these two possibilities (Brent & EVashrub. 1980) (Fig. 1). If t’he Zexd protein directly represses a particular din gene. then removal of lexlg protein should be a sufficient condition for expression. Under normal inducing conditions the Zexil protein is removed by the recA proteasr. However. if the lexd protein has been inactivated by mutation, then recA protease should no longer be necessary to activate the gene. Thus. cells that lack both rrc.-l and lrr3 proteins should produce high levels of P-galactosidase, If, on t,he othtlr hand, a din gene is repressed by a reed-sensitive repressor other than the lexd protein, then the inactivation of t,he Zex;L protein would not necessarily lead to din expression. In a recA mutant, large amounts of non-functional recA protein would be produced, but because the act’ual din repressor could not be cleaved, the gene would not be expressed. In this case, cells lacking both recil and 1rxA proteins should produce low levels of fi-galactosidase.

Thus. in order to distinguish between t)hese two possible situations. \ve

450

80 t

c d e A

600

o b c d e f

r -

180

I20

60

LJllll O cl b cd ef

constructed a set of dir/&w fusion strains in which botjh thr. rwLl anti 1ps.i protciw had been inactivated by mutation. Functional /rx&-l protein MXS removed 1)). introducing a lexA mutation called ,<pr. spr alleles are null mutations of thr Ip.c,, 1 gene since lex;1 amber mutations are SPY alleles (Pawlli cJf r/l.. 1979) and NY’ havc~ recently generated spr alleles by inserting t,hrb t,ransposon Tn:j ill t hr /e.r.-I g~rw (J. H. Krueger &I G. (‘. Walker, unpublished results). As shon-11 in b’igurc~ I, r~vet~~~ strain produces high levels of fi-galac,tosidaw. The finding t,hat remova. of 1r.c. I protein makes rer.4 function dispenwble is inconsixtc~~t wit.h thwcl lwing a I-W.-~ sensit,ive dir/ gene repressor other than the Ip.r.4 prottain. as ~~41 as with a 1uow complicated model that was proposed earlier (\Vitkin. 1976). and strongly suggesth that the ZexA protein directly represnes ewh dir/ gem’.

The conclusion that the IPXA protein directly repressw each rl& gene is Ilot tht only possible interpretation of the genetic st,udies. For example. the data do not rule out the possibility that the lexd protein represses a gene that codes for au activator of din transcription. It was therefore imporbant t,o obt.ain dirwt evitfcnc:r that the Z~x,l pro&in int,eraets with t.hese genes. To do this. KC’ ~loncd thr,

0 t

lac z.

din:: Mud

AP’ HE

+ 0

pf3R322

1

Clone Bum. frogmenis Isolate Lac+ ironsformonts 1

i Digest with HkdEI

Circularize lsolote Lot- tronsformonts 1

AP’ e P B

H

FIG:. 2. (‘onstrwtion rend strwturrs of plasmids carrying the din.4 and dir[K regulatory regions. Tht restriction site+ indicated on the integrated Mud phagr wre determined by M. O’Conner (personal commnrlication). I’lasmids pGWSI0 and p(:R:Sl 1 varry the di~rrl regulatory region, and pQW520 and p(:W521 carry th<> dinB regula.tory regions. Strwturrs of plasmids were determined by standard 1t,st,rict.ion anal,vsis as outlined in the Figurv.

regulatory regions of two dirr genes and determined the effect of purified lr.rA protjein on their transcription ilz vitro.

(c) CZoning of the dinA and dinB regulutory regions

To clone the din/l and dinB fusions, we took advantage of a Ra,mHI site located within the Mud phage about 4 kb downstream from the 3’ end of the 1ac.Z gene. So additional BarnHI sites are located upst,ream in the Mud transcripbional unit. If, in addition, no recognition sites occurred between the end of the integrated Mud phage and the disc promoters. t,hen a single chromosomal RamHI fragment would be expected to contain the din-lac operon fusion.

~‘hromosomal RamHI fragments from the din-4 and din/l fusion strains were cloned int,o t’he RarnHI site of pBR322. and the ligation mixture was transformed intjo strain G\VlOOO. the la,c- parent of the din fusion strains (Fig. 2). Lac+ transformants were isolated from selective plates containing a lactose indicator. In order to determine whether the cloned inserts carried the din regulatory regions. the transformants were tested for u.v. inducibility of fi-galactosidase. In both cases. the transformants showed inducible fl-galactosidase expression (not shown). u.v. inducibility is not a general propert’y of cloned fusions. simr Lac+ transformants that did not show induction were obtained from other experiments.

Preliminary restriction maps of the cloned dinA and d&B fusions are shown in Figure 3. The regulatory regions were subcloned by deleting t,he sequences derived

452

d;nA

Barn p - ---__ --------D Hhd III

I I I

I I

Bum p--------------p Hind III 1’ *

I I

from the Mud phage as outlined in Figure 2. The dirt.4 subclone contains a 2.8 kl) bacterial fragment carrying the din.4 promotjrr, and the rl/‘nK subclone catrics a 3.1 kb insert fragment.

The approximat)e positions of t)he promoters on these fragmr~nts were drterminrtl by in uih transcription experiments. The 2.8 kb c~;HA insert fragment was isolatrd and transcribed in vitro. When the int’act fragmtntj \\-a~ transcaribrd. a high molecular weight smear was observed. The smear probably resulted from brvaka,ye or slight degradation of a high molecular weight transc*ript’. The sensitivity of thca assay was greatly increased by clearing the insert’ fragment with various rr&ric*tion enzymes before transcription. Aft,er digestion a&h a number of restriction endonucleases, a single smaller transcript, was observrd. A Hinf I digestion of the insert fragment resulted in the synthesis of a t.ranscript of about 130 bases. The individual H&f1 fragments were’ isolated and tra,rrsc.ribed individually. ‘I’hv promotir was found to be located on a 540 base-pair fragment. In order to determine the distance of the din.-l promoter from the site of Mud ins&ion. a Hi&l restriction map was obtained (see Pig. 3). From the location of the 540 bp fragment within the cellular sequences. we conclude that’ the din.4 promoter is likei>, to liv about, 1300 bp upstream from the site of Mud insertion.

The 3.1 kb fragment carrying the dinR regulatory region was found to c(tnt.aitl three R&El1 restriction sites, as shown in Figure 3. These t*hree RsttcUI fragments were purified, digested with various restriction endonuclrasrs. and transcribed. l’ht> only strong promoter carried by any of the insert frapment#s was found to be located on the 1480 base-pair HindIII-B&E11 fragment. Among the insert fragments. this one is closest, to the position of Mud insertion. Again, a single major transcript was

I~.~~lA~:~:-lKDI’~‘IBLE GENES 453

observed. When the Hi+xdIII-RstEII fragment, was cleaved with Tag1 and transcribed in vitro a 400 base-pair runoff transcript was observed. The individual Tag1 fragments were isolated and transcribed in vitro. One of these fragment’s (670 bp) directed the synthesis of this 400 bp transcript. The position of this fragment within the BstEII-Hind111 fragment was determined by labeling the BstEII--Hi?,dIII fragment at one end, and then measuring the sizes of the fragments generated by partial digestion with TaqI. The 670 bp fragment was positioned furthest from the Hind111 site (see Fig. 4), indicating that insertion of t,he *Mud phage occurred about 900 bp downstream from the dinB promoter.

The functions of the dinA, dinB, dinD and dinF gene products are not’ known. Tt is interesting to note t,hat both the dinA : : Mud and dinB : : Mud insertions occurred at a distance of about 1 kb from t,he promoters of t,hese genes. It is possible that the din/l and dinB fusion strains do not display striking mutant phenotypes because the insert)ions in t,hese genes do not result in loss of their functions. The availability of cloned sequences should facilitate the construction of additional diw21 and dinI? mutations that might reveal the biological roles of these genes.

din A-

amp -

PI{: 4. Inhibition of transcription of the di?z;1 and dir/B grnrs by purified lex.4 protein. Fragments hearing the diu.4 and dirrR promoters were isolated and transcribed as described in thr text (and in Materials and Methods). A pRR322 HindlII/EcoRI fragment carrying the fi-lactamase promoter was also isolatrd and included in the reaction mixtures. DNA was present at 1 x 1O-9 M in the transcription reactions. and lrsd protrin was omitted (-) or included ( + ) at 5 x lo- M. Similar results were obtained with concentrations of Zexd protein as low as 2 x lo-* w. The concentration of functional l~zd protein present in thr react.ions may be overestimated. as the perwntagr of active lez.4 protein in thP preparation is unknown. amp. ampicillin resistance genr.

Uninduced ccl Is

dinA d;“Fn

/exAk

dinD

DNA damage

DNA repair

SOS-induced cells

(e) In vitro transcrlptio~ oj’&hr din-4 und dinH yews

The effect of lrxrl protein on the in vitro transcription of the rliurl and dinH gcws is shown in Figure 1. ln this experiment, fragments carrying the died or dir~I1 promoters were present in reaction mixt,ures that, also cont~aincd a Hilt,dII-bYoK I

I)A,\IA(;E-INDIT(‘IH1,E: (:ESE:$ 455

fragment from pBR322 containing the 5’ portion of the ampicillin resistance gene. Both DNA fragments were present at relatively low concentrations (1 nmol), to avoid perturbing t’he concentration of free protein by its binding to DNA. When purified ZexA protein (1 x 10-’ M) is pre-equilibrated with the DNA before addition of RNA polymerase and t’riphosphates, transcription from the dinA and dinB promoters is blocked. Concentrations of ZexA protein as low as 2 x 10-’ M

effectively inhibit transcription (data not shown; lower concentrations were not tested). The effect of 1exA protein is specific for transcription of the dinA and d&B promoters, because transcription of the fi-lactamase gene is unaffected. These results confirm that the din.4 and din B genes are direct’ly repressed by 1exA protein.

4. Discussion

We have previously identified a set of genes, called din genes, whose expression is stimulated by agents that induce the SOS response (Kenyon bz Walker, 1981). The results presented in this paper indicate that each of these genes is directly repressed by the same repressor, the ZexA protein. When this protein is genetically removed from cells, the rate of transcript’ion of each gene increases dramatically. In the absence of the ZexA protein, the recA protease is no longer necessary for their expression. Furthermore, purified Zex;l protein inhibits t’he transcription of at least two of these genes in vitro.

By genetic experiments similar to those described here, we have previously obt,ained evidence suggesting that the damage-inducible genes uvrA and umuC whose products function in DNA repair and mutagenesis, as well as the ZexA gene itself. are also directly repressed by the ZexA protein (Kenyon & Walker, 1981: Bagg et al., 1981; Brent Br Ptashne. 1980). Furthermore, we have shown that the l~x=I prot’ein binds to specific operator sequences near the promoter of its own gene and the rp~A gene (Brent 8: Ptashne, 1981), and the protein has been shown to bind to operator sites near the promoters of the u~:rA and /l~rR genes (A. Sancar. unpublished data; W. D. Rupp, personal communication). The protein also appears (by genetic criteria) to repress the s&4 (Huisman & D’Ari, 1981) and himA (H. Miller et al., 1981) genes, whose products function in filamentation and site- specific recombination, respectively.

Figure 5 outlines the basic strategy by which the bacterial cell appears to induce (ho-ordinately the collection of SOS functions in response to DNA damage. In undamaged cells the ZexA protein binds to the regulatory regions of more than 10 (Lellular genes, and inhibits their transcription. When t)he cell’s DNA is damaged, the rrc;1 prot,ein acquires protease act’ivity and begins to cleave molecules of ZexA repressor. As the level of lexd protein falls. the various genes are derepressed, and their products are synthesized. These products participate in a set of metabolic processes. some of which are physiologically unrelated, and which are recognized as the various SO8 functions.

In the screen for mitomycin C-regulated din-lac fusions, 10 independent Mud insert,ions mapping within five din transcription units were obtained (Kenyon PL Walker. 1981). These din genes were identified solely on the basis of their inducibility by mitomycin C, with no requirement that they be controlled by the

456 (‘. .I. KESYON 87’ .-I/,

recA and lex;l proteins, or that they function in t,he SOS response. Yet each of thaw genes appears by genetic (and in some cases biochemical) criteria, to be repressed by the ZexA protein. The finding suggests that if addit,iona,l mit,omycin C-indwiblv genes exist that are not repressed by t,he ler.4 protein. then their frequency is probably much lower than the frequency of IPr-repressed genes.

-4 number of observations suggest that) wrt)ain fine-tuning mechanisms olwrate within t,he framework depict,ed in Figuw 5. For c~xampk~. wrtain SOS-indwing treatments (mat)ing unirradiated tc‘- wlls wit)h u.v.-irradiat,ed Hfrs ((:eorgc rt t/l.. 1974) and infecting restrict,ing hosts w&h unmodified I)NA (Dhartnalingarn & Goldberg. 1980)) induce only a subset of the SOS wsponses. One possibk> explanat,ion for this behavior is that it is a (wnw~utww of different binding affinit,ies to different’ operator sites (Kwnt 8~ l’tashne. 19X1 ). ,Idditional observations suggest t,hat not’ all the pa,rameters that infiurntr dig gew t~xprtwion have bern identified. One particularly puzzling finding is the obsrrvat~ion t,ha,t ow of the din genes (dinD) shows a prolonged lag time before the onwt of /3 galartosidase induction at relatively high doses of 11.v. (60 ,I/m2). but is induwtl a.s quickly as the other dill genes at lo\~w doses (I5 .1/m’) (Kt~nyon. 1981 ). The \~a). in which the cell can differentially regulator t’hr expressiorl of grnrs and prowssw ~withi~t this single regulatory network is thtt ventral umwl~ed prol)lrtn.

CVc- r~xprrss our apprwiatiolr t,o 1). liirnt~lnian. K. Zitttt. .I. Hoc*hsc*hirltl. I,. (;uart~trte. Ii. l’errv. S. Mledge. ,J. Krueger, ,J. Szostak, A. Xlitchrll, ,J. Burr and our other colleagues for their helpful discussions and assistance. We t,han k I,. )Vithcrs for hr help in preparittg thv manuscript. This work was supported by grant no. NIH~I-ROI-(:~I28988-02 to (:.(‘.\il’. front the National Institute of (:enetxl Medical Scienw and It>- grant no. l”c?rl-81-10168 to 11.1’. from the National Science Foundation. (‘.,I .K was supported by a .Johnsott and .Johnsorr A&w)c-iated Industries Fellowship for part of t,his work. (i.(‘.\I’. was a Rita Allen Scholat,.

RFPFRFN(‘l& 1 i 1 11

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Edited by S. Brenner