Transcriptional control of the uvrD gene of Escherichia coli

Gene, 25 (1983) 309-316 Elsevier

309

GENE 882

Transcriptional control of the m,rD gene of Escherichia coli (Mu phage fusions; DNA helicase II; operon fusion; SOS response; chromosome mobilisation; DNA damage-inducible loci)

Helen M. Arthur and Philip B. Eastlake Department of Biochemistry, University of Newcastle upon Tyne, Newcastle upon 7~yne, NE1 7RU (U.K.) Tel. (0632) 328511 ext. 2739 (Received June 13th, 1983) (Revision received July 15th, 1983) (Accepted July 18th, 1983)

SUMMARY

Transcription of the uvrD gene of Escherichia cog was studied using the Mud(Aprlac) gene fusion technique of Casadaban and Cohen [Prec. Natl. Acad. Sci. USA 76 (1979) 4530-4533]. Strains were isolated with Mud(Apqac) inserted in both orientations and chromosome mobilisation experiments showed that transcription of uvrD was from ilvD towards metE. Constitutive expression of uvrD was approximately equivalent to 3000 protein molecules per cell. This level increased 1.5-fold following treatment with DNA damaging agents, an increase which was regulated by the recA and lexA genes. In addition, the constitutive expression of uvrD was reduced in strains containing either the recA 56 mutation or a multi-copy plasmid carrying lexA +. These results indicate that uvrD is an SOS-inducible gene.

INTRODUCTION

The expression of a number of genes in E. cog has been found to be under the control of a common regulatory system known as the "SOS response" (Little and Mount, 1982). The available evidence supports the model that LexA protein is the represser of several SOS genes and that following the inducing signal, which may be a block in DNA replication or DNA damage, LexA protein is proteolytically cleaved by the RecA protein resulting in the induction of the SOS genes. Transcriptional control of several DNA damage-inducible (din) loci including uvrA, Abbreviations: Ap, ampieillin; din, DNA damage-inducibleloci; Mud, Mul defective phage; Nal, nalidixie acid; SDS, sodium dodecyl sulfate; UV, ultraviolet light. 0378-1119/83/$03.00 © 1983 Elsevier Science Publishers

uvrB, umuC, himA, sfi4 (reviewed by Little and Mount, 1982) and rue (Shurvinton and Lloyd, 1982) has been studied using the Mud(Aprlae) phage of Casadaban and Cohen (1979) which allows fusion of operons to the iacZ structural gone. The uvrD gene of E. cell is known to be involved in DNA repair (Van Sluis et al., 1974; Knmmerle and Masker, 1980; Rothman and Clark, 1977; Rothman, 1978), the fidelity of replication (Smirnov et al., 1972; Siegel, 1973a,b), genetic recombination (Horii and Clark, 1973; Arthur and Lloyd, 1980; Howard-Flanders and Bardwell, 1981), mismatch repair (Nevers and Spatz, 1975) and transposon excision (Lundblad and Kleekner, 1982). More recently the UvrD product has been identified as a protein of Mr 73-76 x 10~ (Arthur et al., 1982.; Maples and Kushner, 1982; Oeda et al., 1982). It has

310

been purified and shown to be identical to DNA helicase 11 (Hickson et al., 1983), a previously well characterized protein which has a probable role in DNA replication (glinkert et al., 1980; Kulm and Abdel-Monem, 1982). As UvrD protein plays an important role in DNA repair, we were interested to know whether its synthesis was inducible by DNA damage in a similar way to the UvrA and UvrB proteins 0Fogliano and Schendel, 1981; Kenyon and Walker, 1981; Sancar et al., 1982) or whether it was under some other regulatory control. To approach this question we isolated fusions of Mud(Ap'lac) in the uvrD gene such that the synthesis of~galactosidase was under the control of the uwD promoter. In this paper we show that uwD is one ofthe SOS genes. It has a high constitutive level of expression but shows a small /exA/recA-reguLated induction following treatment with DNA d a m a .olng agents.

METHODS

1976). Pl transduction followed the procedure of Miller (1972). (b) lsolatim of Mud(Ap'la¢) f m i e m Colonies of N 1972 containing random insertions of Mud(Ap~lac) were isolated using the procedure of Casadaban and Cohen (1979). Phage lysates were prepared by thramal induction of strain MALl03 and used to infect the strain N1972. Cells resistant to 40 pg/ml ampicillin were selected at 3 0 ° C . (c) M e u a r e m ~ t o f ~-gaLactosidase expression The level of ~-galactosidase was measured essentially by the method of Miller (1972). Cells were grown in minimal medium at 30°C. Samples of 0.1 ml were diluted in 2.9 ml M56/2 and lysed by the addition of 50/d 0 . 1 ~ SDS and 10/d chloroform prior to assaying j~-galactosidase activity. To induce the cells mitomycin C (final concentration I pg/ml) or nalidixic acid (100pg/ml) was added to the growing cells and for UV induction cells were irradiated with 10 J/m 2.

~ Strata ~ C'eromeseme mebinsatim The E. coli strains used are listed in Table I. The methods for strain construction and Elfr crosses have been descn'bed previously (Lloyd and Low,

Derivatives of Mud(Ap~/ac) filsion strains which carried F'128 p m A B + lac + were grown in LB to

TABLE ! E. cob K-12 strains Strain

~

Source or reference

MALl03 F_5014 N1972 HA1485 HA1486

F- Muasdl(Al//ac) Mutts d(pvoAB4nc}Xlll rps/. F'128 proAB + /ac+/d(proAB4ac)Xlll 06-I ma/-24 spc-12supES0 F- k~-29 metE90 rp~LlTl na/A 19 d(/m~IB-/ac)Xlll As N1972 but mrD: :Mud(Al//ac) As N1972 but mr/): :Mud(Apr/ac)

HAIl81 HAI482 itAII87

As HA!485 but Met + As HAI486 but Met+ As HAl181 but F'128

Casadaban and Cohen, 1979 R.G. Lloyd R.G. Lloyd This work This work KL226 x HAI485-, Met*(t~J.)

HA1484

As HA1482 but F'128

F_.5014x HA1482--. Pro+(r/uL)

HAI488

As HAI485 but n.cA56 F- /h,DI88 metE~ nlt~19605pmB48 tp~/..171 ~

PI.NI425 x HAI485--.Tet" Laboratorystrain

F- pmB48 /ds-29 frpA9605 a~pl-/~./3 /acZll8 q~sLl71 na/Al9 mrD210 As N1246 but m y D F- Od-I Ms-4/m~12 Otr-I/cuB6ara~14/acYl ga/X2 nu/-lxy/-5o/: :Tnl0 rec,456 rpsL31 ropE44 Hfr(Cavalli)rdA I ww122

Arthur and Lloyd. 1980

HAI487 N1246 NI247 N!425

÷

KL226 x HAi486-+Met+(~) E5014 x 1tA!i81 -+ Pro+(~L)

Arthur and Lloyd, 1980 R.G. Lloyd

ILB. Low

311

2 x l0 s cells/mi and mixed in the ratio I to 5 with a Mu(c ÷) lysogen of the recipient strain HA1487 (proB- ilvD- metE- R~). Samples of the mating mixture were taken after 30 rain to measure the frequency of episome transfer by the number of Pro ÷ Rif repliconants and after 90 min to measure the frequency of chromosomal mobilisation by the number of Met ÷ Rif or llv + RiP" recombinants.

RESULTS

O

•

8

10-1

e-

9 u m Lz.

(a) Characterisation of Mud(Aprla¢) fusions to the mwD gene Ap" colonies were screened for sensitivity to UV light and for an increased frequency of spontaneous mutations to rifampicin resistance. This is the phenotype expected of a uwD mutant and two such strains were found from 2000 colonies screened. One strain HA1485 had the Mud(Ap'/ac) fusion in the correct orientation for transcription of/~galactosidase and had a Lac + phenotype; the other strain HA1486 contained the insert in the opposite orientation and was L a c - . Hfr crosses and PI transductions were used to map the positions of the Mud(Aprlac) insertions and to verify that each strain carried only a single Mu prophage. Both insertions were found to lie between / / ~ and metE and showed 34~ linkage to metE in PI transduction, which agrees approximately with the linkage frequency of uvrD210 (Arthur and Lloyd, 1980). Complementation studies using pHMA3(u~'D ÷ ) (Arthur et al., 1982) showed that the UV sensitive and mutator phenotypes conferred by the Mud(Aprlac) inserts were fully complemented in the presence of a low copy number pIR.cmid carrying the wild-type urrD gene, priMA3, but not by a derivative of this plasmid in which the urrD gene had been inactivated by Tnl000 (Fig. I). Oh) Cemslitefive levels of UvrD I[WOtein

The uwD::Mud(Ap'lac) strain, HA1485, had a high constitutive level of p.galactosidase activity of between 120 and 150 unlts/A6,o (Fig. 2). A similar although marginally lower level (100 to 130 units/ Aeoo) was found in HA1485 harbouring priMA3, a

~ 10"~

-\:

10"

10"0

&

l

2

I

l

4

6

uv ~

j/m2

~

l

10

Fig. !. UV ramsiti~ly otm~rD: : Mud(Apqa¢) slrahm. C.,dls~ grown in LB to 2 x 10z ce]~jmL Appropriate ¢h~limm were

plated onto LB asar and irr-ai.ted with UV lizhL The retains medw~¢ HAS4S5(A), HAt4S6 (A~ HAt4SS barbowin8pZ~mid pEIMA3 (~rD+)(O), HAI486 ~ pEIMA3 0nrD ÷ ) (O), HA!485 harbomins pl-iMA3uwD: :Tn/000 ( n ) and HA!486 baxbouring p ~ D : :To/000 ( i ~ .

low copy number plasmid carrying uwD ÷. If the assumption is made that the RNA and protein products of the uvrD and/acZ genes have .¢indlar half lives, t h ~

figth'~ Can b e u s e d t o calcnlate t h e

approximate concentration of UvrD protein in the celL When the/ac operon is fully induced in a wgdtype strain the level of p - ~ , t , ~ s i d a s e activity is 1000 m~,s/A6oo ~ , 1972) which corresponds to approx. 20000 monoma's of,8.gd,,gtoslda~ per (Cohn, 1957). Thereforc, the constitutive level of /3-plactosidase activity in the uwD::Mud(Ap'/ac) fusion strain corresponds to between 2400 and 3000 molecules of UvrD protein per celL This is in rough agreement with the 5000 molecules of D N A hdicase II per Hell reported by Kfinkm et aL (1980). The uwD::Mud(Ap~/ac) strain HA1486 in which

312 b

a

/

240

210

.f"

./

o 180 0 cD < "" 150

/& &

w r-

A

12G - - &

/~

/ @

A

N

^

t~

,-~ . - - - - - - -

0 ~

9 2

9 3

o~

g

? 4

, 5

0

6G

30 0

0

u 1

. 6

[]

7 0 Time (hours)

, 1

, 2

, 3

, 4

, 5

Fi&2. p-galactosidase synthesis in u w D : : M u d ( A p f f a c ) fusion strains. The enzyme units have been defined as 1000 x (A420-135 x Asse)/t x v x Aeoo where t = time ofthe reaction in minutes and v = volume ofculture used in the assay, in ml (Miller, 19.72). DNA damaging agents were added i h a t t e r the start of the experiment as indicated by the arrow. (a)HAI485 (A), HA1485+I/tg/ml mitomycin C ( & ) , HA1488(O), H A I 4 8 8 + l / ~ m l mitomycin C ( O ) , HA1486(1"]). (b) HAI485 (A), HA1485 + 10 J/m 2 UV light ( ~ ) , HA1485 + 100 pK/ml nalidixic acid (A), HA1485 harbouring pPE24(lexA ÷ ) with ( 0 ) or without ( O ) addition of I pg/ml mitomycin (2.

the lac gene is inserted in the "wrong" orientation with respect to the uvrD promoter had a constitutive level of ~q-galactosidase activity of only 1 u n i t / A ~ o (Fig. 2a).

(e) Inducible expression of the uvrD gene The level of ~-galactosidase activity in HA1485 increased 1.5-fold over the level in the untreated control 4 halter treatment with 1 pg/ml mitomycin C (Fig. 2a). To verify that this small increase was sisnificant the experiment was repeated 6 times and on each occasion the same induction was found. A similar result was observed when 100 pg/ml nalidixic acid was used as the inducing treatment (Fig. 2b) and in both cases a time lag of approx. 1 h was observed prior to induction. A smaller induction of 1.3-fold occurred after the cells were irradiated with 10 J/m a UV light. When either the recA56 mutation or a multicopy plasmid carrying lexA + (pPE24; Emmerson et al.,

1981) was introduced into HA1485 the constitutive level of ~galactosidase was reduced to between 75 and 105 enzyme units/A6oo (Fig. 2, a and b). In addition, the presence of plexA + plasmid or the recA56 mutation completely suppressed the inducible expression of uvrD by mitomycin C (Fig. 2, a and b) indicating that the induction was regulated by the recA and lexA genes. (d) Effect of nalidixic acid Nalidixic acid gave the highest induction of uvrD expression, a 1.7-fold increase after 4 h. A high concentration (100 pg/ml) was used because the Mud(Aprlac) fusion strain carries a nald - mutation and is therefore resistant to low concentrations of nalidixic acid. However, we have observed that nabt strains become sensitised to nalidixic acid when a uvrD mutation is introduced. Fig. 3 shows that the uvrD210 mutation in the strain N1246 (Arthur and Lloyd, 1980) and the uvrD:'Mud(Aprlac) mutation

313

TABLE 11 ~

o

Chromosome mobilisation frequencies of ih,D and m e t E

in

strains HAI484 and H A l 187. The frequency ofepisome transfer is measured by the number of Pro + Rift replieonants recovered. The procedure used is described in M A T E R I A L S A N D METH O D S , section d. Cross

1 ~

':\

1o 3

Exconjugants

Frequency

selected

(No./ml) a

HAI484

Pro + Rift

1.4 x l0 s

x

Met + Rifr

1.7 x 103

HAI487

llv ÷ Rift

1.2 x 10a

HAIl87 x HAI487

Pro + Rifr Met + Rif t llv + Rifr

6.7 x 10"1 2.7 x 103 6.1 x l02

Ratio

l l v + / M e t + = 7.1

M e t + / l l v ÷ = 4.4

Correction was made for the frequency of spontaneous Rift mutants in HA1484 and HA!187.

"\\.

\.',,.

10 4

10.-5 0

! 50

Nalidixic acid

i 100

i 150

(pg/ml)

Fig. 3. Nalidixic acid survival of NI246 (uvrD210 nal-19)(O), NI247 (uvrD + ha/-19) (O), HA1485 (uvrD : : M u d ( A p ~ l a c ) n a l -19) (&), NI972 (uvrD + ha/-19) (A). Cells were grown in LB to 2 x l0 s and appropriate dilutions were spread onto LB agar containing different concentrations of nalidixic acid. Survivors were counted after 36 h incubation at 30°C.

in HA1485 dramatically increase the sensitivity of these naIA - strains. This suggests that there is some, as yet unidentified, interaction between DNA helicase II and DNA gyrase.

transfer is known (Low, 1972). Thus, recombination between the homologous lacZ of the episome and lacZ in the Mud(Aprlac) insert will result in a high frequency of transfer of either metE or ilvD depending upon the orientation of the Mud(Aprlac) insert (Fig. 4). The Lac ÷ strain HA1485 mobilised metE at a 4.4-fold higher frequency than ilvD, whereas the Lac - strain HA 1486 mobilised ilvD at a 7-fold higher frequency than metE (Table II). The two Mud(mprlac) fusion strains mobilised the chromosome in opposite directions confLrming that they carry Mud(Aprlac) inserts in opposite orientations. The direction of mobilisation in the Lac ÷ strain, HA 1485, indicated that the direction of transcription of uvrD is from ih,D towards metE (Fig. 4).

DISCUSSION

(e) Determination of the direction of transcription using chromosomal mobilisation The Mud(Ap~lac) fusions in uvrD were used to determine the direction of transcription of uvrD with respect to the neighbouring genes ilvD and metE. This was achieved by using derivatives of the Mud(Ap~lac) fusion strains which carded F' 128proAB ÷lac ÷ and determining the direction of mobilisation of the host chromosome into a recipient strain, HA1487. The orientation of lacZ on the episome with respect to the direction of episome

Our results indicate that expression of uvrD is inducible following damage to DNA by UV-light, mitomycin C or nalidixic acid and is under recA/lexA control. This conclusion is supported by the DNA sequence analysis presented in the accompanying paper (Finch and Emmerson, 1983) which reveals a potential LexA binding site in the control region of the uvrD gene. Sequence analysis also indicates that there may be a second promoter downstream of the LexA binding site which might contribute to the high constitutive level of UvrD expression observed even

314

ro~

Mud_lac Z

:"- Y

\

t \

I \

/ \

/ \

,,u"'v ''+ i

uvrD

/

~~

/ .~

~'

t

met E + I

Fig. 4. Diagram to show the orientation of the Mud(Aprlac) insert in HA1485. The direction of transcription of lacZ is indicated by the wavy line, and homologous recombination between the episome F' 128 and the Mud(Apqac) insert by dotted lines. In this case mete would be mobilised at a higher frequency than #yD.

in the presence of a multi-copy plasmid carrying

reeL152 nal÷ strain was more sensitive to nalidixic

lexA ÷

acid than a wild-type strain. Both of these results suggest that there is some interaction between UvrD protein and DNA gyrase. We estimate that the constitutive level of UvrD protein in E. coli is approx. 2400 to 3000 molecules per cell. One explanation of this high level could be that the measurements were made in a uvrD- strain, uvrD having been inactivated by insertion of Mud(Apqac), and that DNA lesions which are thought to accumulate in uvrD- strains lead to constitutive SOS induction. However, a similar level of uvrD expression was observed in the presence of a low copy number plasmid carrying uvrD ÷. Therefore, if uvrD- mutants accumulate DNA lesions, these alone are not sufficient to fully induce SOS expression of the uvrD gene. The high constitutive level of UvrD protein would be compatible with evidence suggesting a role in DNA ~:eplication (Klinkert et al., 1980; Kuhn and Abdel-Monem, 1982) whereas the induced protein may be required for DNA repair. The coneentrarion of UvrD protein appears to be critical to the cell. For example, a mulri-copy plasmid carrying uvrD ÷ sensirises wild-type strains to UV light (Oeda et al., 1981; Maples and Kushner, 1982) suggesting that too high

Using a similar approach to ours, Siegel, E.C. (personal communication) has also found a lexA/ reed-regulated induction of uvrD expression and Kimura et al. (1983) have detected a recA-dependent 4--6-fold incre&s~ in the level of a DNA-dependent ATPase. corresponding to the UvrD protein following mitomycin C or. nalidixie acid treatment of wildtype cells. The isolation of two Mud(Apqac) fusions in opposite orientations in uvrD permitted the direction of transcription to be determined with respect to the neighbouring genes metE and ilvD. Chromosomal mobilisation experiments show that transcription of uvrD occurs from ilvD towards metE. This agrees with the conclusion that uvrD is transcribed towards eorA (Maples and Kushner, 1982) assuming that the gene order is ilv uvrD corA mete (Kushner et al., 1978.). Under the conditions used in our experiments nalidixic acid proved the most effective inducer of u,vrD expression.: However, we have observed that Nalr(na/A) strains become Nal" when a uvrD mutation is intt.oduced. Similarly McDaniel et al. (1978) have. reported that under certain conditions a

315 a level o f u v r D protein m a y p e r t u r b D N A mechanisms.

repair

ACKNOWLEDGEMENTS W e are grateful to D r . R . G . L l o y d for b a c t e r i a l strains a n d the M e d i c a l R e s e a r c h C o u n c i l for f'mancial s u p p o r t .

REFERENCES Arthur, H.M. and Lloyd, R.G.: Hyper-recombination in uvrD mutants ofEscherichia coliK-12. Mol. Gen. Genet. 180 (1980) 185-191. Arthur, H.M., Bramhill, D., Eastlake, P.B. and Emmerson, P.T.: Cloning of the uvrD gene of E. coil and identification of the product. Gone 19 (1982) 285-295. Casadaban, M.J. and Cohen, S.N.: Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional eontr01 sequences. Proc. Natl. Aead. Sci. USA 76 (1979) 4530--4533. Cohn, M.: Contributions of studies on the ~-gaiactosidase of Escherichia coli to our understanding of enzyme synthesis. Bacteriol. Rev. 21 (1957) 140-168. Emmerson, P.T., Hickson, I.D., Gordon, R.L. and Tomkinson, A.E.: Cloning ofrecA + and lexA + and some of their mutant alleles: an investigation of their mutual interaction, in Seeberg, E. and Kleppe, K. (Eds.), Chromosome Damage and Repair. Plenum, New York, 1981, pp. 281-285. Finch, P. and Emmerson, P.T.: Nueleotide sequence of the regulatory region of the uvrD gone ofE. coli. Gene 25 (1983) 317-323. Fogliano, M. and Schendel, P.F.: Evidence for the inducibility of the uvrB operon. Nature 289 (1981) 196-198. Hickson, I.D., Arthur, H.M., Bramhill, D. and Emmerson, P.T.: The E. coil uvrD gone product is DNA helicase II. Mol. Gen. Genet. 190 (1983) 265-270. Horii, Z.-I. and Clark, A.J.: Genetic analysis of the Reef pathway to genetic recombination in Escherichia coli K-12: Isolation and characterization of mutants. J. Mol. Biol. 80 (1973) 327-344. Howard-Flanders, P. and BardweU, E.: Effects of ree821, reeF143, and uvrD 152 on recombination in lambda baeteriophage-prophage and Hfr by F - crosses. J. Bacteriol. 148 (1981) 739-743. Kenyon, C.J. and Walker, G.C.: Expression of the E. coil uvrA gone is inducible. Nature 289 (1981) 808-810. Kimura, K., Coda, K., Akiyama, M., Horiuchi, T. and Sekiguchi, M.: The uvrD gone of E. coli: Molecular cloning and expression, in Friedberg, E.C. and Bridges, B.R. (Eds.), Cellular Responses to DNA Damage, UCLA Symposia on Molecular

and Cellular Biology, New Series, Vol. 11. Liss, New York, 1983, in press. Klinkert, M.-Q., Klein, A. and Abdel-Monem, M.: Studies on the functions ofDNA helicase I and DNA helicase II ofE. coli. J. Biol. Chem. 255 (1980) 9746-9752. Ktlmmerle, N.B. and Masker, W.E.: Effect of the uvrD mutation on excision repair. J. Bacteriol. 142 (1980) 535-546. Kuhn, B. and Abdel-Monem, M.: DNA synthesis at a fork in the presence of DNA helicases. Eur. J. Biochem. 125 (1982) 63-68. Kushner, S.R., Shepherd, J., Edwards, G. and Maples, V.F.: uvrD, uvrE and reeL represent a single gone, in Hanawait, P.C., Friedberg, E.C. and Fox, C.F. (Eds.), DNA Repair Mechanisms. Academic Press, New York, 1978, pp. 251-254. Little, J.W. and Mount, D.W.: The SOS regulatory system of E. coll. Cell 29 (1982) 11-22. Lloyd, R.G. and Low, B.: Some genetic consequences of changes in the level of recA gane function in Escherichia coil K-12. Genetics 84 (1976) 675-695. Low, K.B.: Escherichia coil K-12 F-prime factors old and new. Bacteriol. Rev. 36 (1972) 587-607. Landblad, V.L. and Kleekner, N.: Mutants of E. coil K-12 which affect excision of transposon Tnl0, in Lemontt, J.F. and Generoso, W.M. (Eds.), Molecular and Cellular Mechanisms of Mutagenesis. Plenum, New York, 1982, pp. 245-258. Maples, V.F. and Kushner, S.R.: DNA repair in E. coil: Identification of the uvrD gone product. Prec. Natl. Acad. Sci. USA 79 (1982) 5616-5620. McDaniel, L.S., Rogers, L.H. and Hill, W.E.: Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid. J. Bacteriol. 134 (1978) 1195-1198. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972, p. 354. Nevers, P. and Spatz, H.C.: Escherichia coli mutants uvrD and uvrE deficient in gone conversion of 2-heteroduplexes. MoL Gen. Genet. 139 (1975) 233-243. Oeda, K., Horiuchi, T. and Sekiguchi, M.: Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency. Mol. Gen. Genet. t84 (1981) 191-199. Coda, K., Horiuchi, T. and Sekiguchi, M.: The uvrD gane of E. coil encodes a DNA-dependant ATPase. Nature 298 (1982) 98-100. Rothman, R.H.: Dimer excision and repair replication patch size in a reeL mutant of Escherichia coil K-12. J. Bacteriol. 136 (1978) A.~.A448. Rothman, R.H. and Clark, A.J.: Defective excision and postreplication repair of UV-damaged DNA in a reeL mutant strain orE. coil K-12. Mol. Gen. Genet. 155 (1977) 267-277. Sancar, G.B., Sancar, A., Little, J.W. and Rupp, W.D.: The u~rB gone of Escherichia coil has both/erA-repressed and lexAindependent promoters. Cell 28 (1982) 523-530. Shurvinton, C.E. and Lloyd, R.G.: Damage to DNA induces expression of the ruvgene ofEscherichia coli. MoL Gem. Genet. 185 (1982) 352-355. Siegel, E.C.: Ultraviolet-sensitive mutator strain of Escheticbia coil K-12. J. Bacteriol. 113 (1973a) 145-160.

316 Siegel, E.C.: Ultraviolet-sensitive mutator mutU4 of Escherichia coil inviable with polA. J. Bacteriol. 113 (1973b) 161-166. Smirnov, G.B., Filkova, E.V. and Skavronskaya, A.G.: The mutator property of uvr502 mutation affecting UV sensitivity of Escherichia coll. Mol. Gen. Genet. 118 (1972) 51-56.

Van Sluis, C.A., Mattern, I.E. and Paterson, M.C.: Properties of uvrE mutants of Escherichia coli K-12, I. Effects of UV irradiation on DNA metabolism. Mutation Res. 25 (1974) 273-279. Communicated by R.W. Davies.