Mutation Research, 254 (1991) 27-35 DNA Repair Elsevier
27
MUTDNA 06404
Construction of Escherichia coli K12 phr deletion and insertion mutants by gene replacement Susumu Akasaka i and Kazuo Yamamoto 2 +Division of Industrial Health, Osaka Prefectural Institute of Public Health, Higashinari, Osaka 537 and 2 Biological Institute, Faculty of Science, Tohoku University, Sendai 980 (Japan)
(Received 16 January 1990) (Revision received 11 May 1990) (Accepted 16 May 1990)
Keywords: phr replacement mutants; DNA photolyase; Pyrimidine dimers; Suppressor mutations
Summary We replaced an Escherichia coli phr gene by a 1.4-kb fragment of DNA coding for resistance to chloramphenicol. Characterization of 2 deletions (phr-19 and phr-36) and 1 insertion (phr-34) in the phr gene revealed no photoreactivation. Photoreactivation-deficient strains of either recA56 or lexAl(ind-) were more sensitive to UV radiation in the dark than phr-proficient counterparts. The presence of the phr defect in uvrA6 strains increased by 1.5-2-fold his-4(Ochre) to His + mutation induced by ultraviolet light compared to uvrA6 phr ÷ strains, although there was no difference in UV sensitivity between uvrA6 phr ÷ and uvrA6 phr- strains. 30-35% of the His ÷ mutations thus induced were suppressor mutations in uvrA6 phr ÷ and 49-55% in uvrA6 phr- strains. The UV mutagenesis results are consistent with the previous observations that suppressor mutations targeted by a thymine-cytosine pyrimidine dimer are reduced in the dark in cells with amplified DNA photolyase.
Cis-syn cyclobutane dipyrimidine dimers (pyrimidine dimers) are induced in DNA by 254 nm ultraviolet light and are one of the primary lesions responsible for UV-induced carcinogenesis, mutation and cell lethality. Enzymatic photoreactivation, mediated by DNA photolyase which is coded by the phr gene at 16.2 min on the Escherichia coli linkage map (Sancer and Rupert, 1978a; Youngs and Smith, 1978), is one pathway by which these lesions are repaired; pyrimidine dimers are bound by photolyase which subsequently absorbs a pho-
Correspondence: K. Yamamoto, Biology Institute, Faculty of Science, Tohoku University, Sendal 980 (Japan).
ton of near-UV light and utilizes this energy to cleave the cyclobutane ring linking the 2 pyrimidines (Rupert, 1975). In addition to photoreactivation, E. coli possesses other repair pathways by which pyrimidine dimers are removed from DNA or genotoxic effects of these lesions are bypassed. These include excision repair, recombination repair and SOS repair (Walker, 1984). Because pyrimidine dimers in DNA are potential substrates for several repair pathways in the same cell, interactions between components of these pathways must be considered in assessing the contribution of each pathway to repair and damage tolerance. Such a notion was supported by a number of recent studies which
0921-8777/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
28
have demonstrated that, even in the absence of photoreactivating light, bound photolyase plays a repairing role in E. coli in the dark. E. coli recA phr ÷ strains are more resistant to UV-induced cell killing than are recA phr- strains (Yamamoto et al., 1983a; Hays et al., 1984). E. coli photolyaseoverproducing uvr-deficient strains are more UVsensitive than uvr-deficient strains producing normal enzyme levels (Yamamoto et al., 1984). With cells containing amplified amounts of photolyase, mutations targeted at thymine-cytosine pyrimidine dimers (T = C) are greatly reduced or eliminated compared to cells containing normal levels of photolyase (Ruiz-Rubio et al., 1988). The fact that photolyase can bind to pyrimidine dimers and then manifest repair effects in the absence of light relates to the question of how much each of the repair pathways contributes to the observed UV results. A genetic approach to answer this question would use isogenic pairs of phr ÷ and phr- strains and compare the effects of UV radiation. There are phr mutants available: phr-1 (Sancer and Rupert, 1978b), phr-2 (Harm
and Hillebrandt, 1962), A(kdp-gltA)214 (Hays et al., 1984) and A(kdp-gltA)219 (Rhoads et al., 1978). However, since a good selective marker for phr does not exist, P1 phage transduction using these alleles has sometimes encountered difficulty. In this paper we describe how we inserted a selectable marker, resistance to chloramphenicol ( C m r ) , into a phr gene and introduced it into the bacterial chromosome by homologous recombination (Winans et al., 1985). The new alleles can easily be transduced by the P1 phage to construct isogenic pairs of phr ÷ and phr- strains. Analysis of the resulting strains suggests that (i) the photoreactivation-defective phenotypes of these mutants are similar to those of mutants with previously isolated phr-1 and phr-2 genes, (ii) a recA phr + strain is more resistant to UV than a recA phr strain in the dark, (iii) a lexA(ind- ) phr + strain is more UV-resistant than a lexA(ind-) phr strain in the dark, (iv) there are no significant differences in UV sensitivity between uvrA phr ÷ and uvrA phr strains in the dark and (v) uvrA phr- strains are more mutable than uvrA phr + strains. UV
TABLE 1 LIST OF STRAINS Name
Relevant genotype
Reference or source
argE3 his-4 leu-6 proA2 thr-I thi-1 rpsL31 galK2 lacY1 ara-14 xyl-5 mtl-1 supE44 As ABl157 but uvrA6 As KY1836 but phr-36 As KY1836 but phr-19 As KY1836 but phr-34 As ABl157 but phr-36 As ABl157 but srlC300:: TnlO recA56 As KY1220 but srlC300: : TnlO recA56 As ABl157 but malE :: TnlO lexA1 As KY1220 but malE :: TnlO lexA1 purA45 uvrA6 gal-6 As K Y l l 6 but phr-2 gal + As K Y l l 6 but phr-19 As K Y l l 6 but phr-34 As K Y l l 6 but phr-36 recB21 recC22 sbcBl5
Howard-Flanders and Theriot (1965) This work This work This work This work This work Yamamoto et al. (1984) This work This work This work Yamamoto et al. (1983b) Yamamoto et al. (1983b) This work This work This work Kushner et al. (1971)
pBR322 + 20-kb phr fragment pBR322 + 2.8-kb phr fragment As pKY1 but phr:: cat ( = phr-19) As pKY33 but phr :: cat ( = phr-34) As pKY33 but phr:: cat ( = phr-36)
Yamamoto et al. (1983a) Ruiz-Rubio et al. (1988) This work Yamamoto and Bockrath (1989) Yamamoto and Bockrath (1989)
E. coli ABl157 KY1836 KY1226 KY1839 KY1326 KY1220 KY1056 KY1225 KY 1701 KYI221 KYll6 KY136 KY196 KY197 KY 198 JC7623
Plasmid pKY1 pKY33 pKY19 pKY34 pKY36
29 mutagenesis targeted at T = C increases in a uvrA phr- strain compared to a uvrA phr + strain.
growth were carried out under yellow light to prevent uncontrolled photoreactivation.
Materials and methods
Survival and mutation assays After UV irradiation, and photoreactivation if applied, surviving colonies were counted following overnight incubation at 37 ° C on Luria agar plates. His ÷ prototrophic mutants were counted, and the samples were isolated after the plates had been incubated for 3 days at 37 ° C. Mutation frequencies were calculated by subtracting the numbers of spontaneous colonies per plate from the numbers of colonies on plates inoculated with treated cells and then dividing these net numbers by the numbers of viable cells assayed per plate. Since both his-4 and argE3 markers in derivatives of strain ABl157 are ochre nonsense mutations and suppressed by glutamine tRNA ochre suppressor mutations (Kato et al., 1980), one can easily identify His + prototrophic mutants as being back or suppressor mutants. All His ÷ mutants per plate (approximately 100 colonies) were patched on minimal salt agar minus arginine and minus histidine plates. Suppressor mutants were identified as those that were able to demonstrate growth on both plates. Back mutants were those that could grow only on the histidine-free plates.
Bacterial strains and plasmids The bacterial strains and plasmids used in this work are listed in Table 1. P1 phage transduction was used to construct isogenic pairs of strains (Ihara et al., 1985). The constructions of plasmids utilized in this work except pKY19 have been reported previously (Yamamoto et al., 1983b; Yamamoto and Bockrath, 1989). Growth media Cultures to be irradiated were grown in Luria broth. Phosphate buffer (4.5 g KH2PO 4, 4.7 g N a 2 H P O 4 per liter of distilled water, p H 6.8) was used for washing, resuspending and diluting cells. Minimal salt agar medium was made of M56 salt plus 0.35% glucose, 1.5% agar supplemented with thiamine (1 /~g/ml) and the amino acids proline, threonine, leucine, histidine and arginine (each at 100 /Lg/ml). His + prototropic mutants were selected on semi-enriched minimal agar (SEM, minimal salt agar medium plus 0.004% casamino acids and no histidine). Minimal salt agar medium lacking histidine or arginine plates was used for His + mutant differentiation. UV radiation and photoreactivation Overnight, stationary-phase cultures of E. coli strains were washed in phosphate buffer, and resuspended in the buffer at about 5 × 108/ml. Cells in 90 mm diameter glass petri dishes were exposed to 254 nm UV light with constant shaking using 2 15-W germicidal lamps (Matsushita Electric Co.). The fluence rate measured by a Topcon UV dosimeter was 0.66 j / m 2 / s . For photoreactivation, 2 Matsushita daylight fluorescent lamps at 20 cm distance were use as the light source. Polyvinyl chloride plastic (0.2 cm layer) and a glass petri dish lid were placed between the photoreactivation light and the sample aliquots in buffer. That the polyvinyl chloride plastic used in this experiment absorbs wavelengths below 380 nm was established with an ISCO UVIDEC 610E spectrophotometer. All irradiation, subsequent manipulations and
Southern blotting The chromosomal D N A blot was probed with a 1.3-kb MluI-MluI phr fragment of pKY33 which was terminal-labeled with [y-32p]ATP with T4 polynucleotide kinase (Takara). Hybridization was done by the procedure of Southern (1975). Results
Construction of the phr deletion and insertion mutations Plasmid pKY1 and the derivative pKY33 carrying the phr gene were used to receive insertions of a chloramphenicol acetyltransferase (cat) gene derived from pACYC184. Plasmid pKY33 contains a 2.8-kb AccI fragment from pKY1, which in turn carries a 20-kb EcoRI fragment from the E. coli chromosome that includes the phr gene. There are 3 MluI sites in pKY33 and 4 in pKY1. The recessed termini of linear isolates from pKY1 after full digestion with MluI or from pKY33 after
3o partial digestion with Mlul were filled in with K l e n o w polymerization, a n d these were ligated to a 1.4-kb HaelI f r a g m e n t from p A C Y C 1 8 4 (cont a i n i n g cat) that was digested to b l u n t ends with Klenow. Restriction maps were then d e t e r m i n e d of several isolated plasmids that conferred both ampicillin a n d c h l o r a m p h e n i c o l resistance a n d were phr-deficient. This i d e n t i f i e d p l a s m i d s p K Y 1 9 , p K Y 3 4 a n d p K Y 3 6 (Fig. 1A). Plasmid p K Y 1 9 was derived from pKY1. A n 8-kb PstI linear fragment carrying the cat gene within a deletion from the first to the fourth Mlul site, that is 300 b p u p s t r e a m a n d 4 kb d o w n s t r e a m of the phr, could be isolated from pKY19. Plasmids p K Y 3 4 a n d p K Y 3 6 were derived from pKY33. A 4.2-kb AccI linear fragment from p K Y 3 4 a n d a 3.9-kb fragment from p K Y 3 6 carrying the cat insertions could be isolated from p K Y 3 4 a n d pKY36, respectively. The former contained the insertion after the first 88 bp of the p r o t e i n coding sequence of phr. The latter had the cat insertion in place of a deletion between the p r o m o t e r area (300 bp u p s t r e a m of the phr) a n d the 88th bp of the phr sequence. The linear fragments thus isolated c o n t a i n i n g cat insertions were used to transform a c o m p e t e n t recB21 recC22 sbcB15 strain (JC7623) ( K u s h n e r et al., 1971). T r a n s f o r m a n t s were selected for C m r. P1 lysates were m a d e on the J C 7 6 2 3 : : cat transf o r m a n t s a n d were used to transduce a p p r o p r i a t e strains to C m r. The C m r p h e n o t y p e proved to be a p p r o x i m a t e l y 10% c o t r a n s d u c i b l e with gal by P1 t r a n s d u c t i o n , as was expected for the phr locus ( Y o u n g s a n d Smith, 1978). C m r t r a n s d u c t a n t s of a uurA6 phr + strain ( K Y I 1 6 ) were e x a m i n e d by a streak test for phr + or phr a n d were all f o u n d to be phr-deficient. C h r o m o s o m a l D N A blots of a phr + strain a n d 3 CmLderived strains c o n f i r m e d that these cells contained the desired phr deletions or insertions (Fig. 1B). W e refer to the phr replacement alleles as phr-19, phr-34 a n d phr-36 (from pKY19, p K Y 3 4 a n d pKY36, respectively).
U V inactivation and photoreactivation of phr replacement mutants T o d e t e r m i n e whether phr replacement m u t a n t s differed from the previously isolated phr m u t a n t s , the UV sensitivity a n d photoreactivability were
(A)
E
P P I
pK¥1 ', pKY33
E
I
\11--/[
I
ANM
M
"I
r
P P ,., i
pKY19 I
AM~NAP M P
M/HE
I
E
I
I
A
' ivl/H P
~
I
E
,
I
pKY34
pKY36
(B)
123
45
678
-23
9-4~ 9.0- .~.
8.0/ #7-
9.6
6-6
4-4 2.3
Fig. 1. Construction of phr deletions and insertion. (A) pKY1 has a 20 kb-fragment containing the phr gene (solid rectangle) inserted at the EcoRl site in pBR322 (Yamamoto et al., 1983b). pKY33 is constructed from pBR322 with insertion of a 2.8-kb Accl fragment containing the phr gene from pKY1 via pBG6 (Ruiz-Rubio et al., 1988). We cloned a blunted 1.4-kb Haell fragment containing the (.at gene (hatched double line), derived from pACYC184, into blunted Mlul sites into pKY33 to generate pKY34 and pKY36 (Yamamoto and Bockrath, 1989), and into pKY1 to generate pKY19, pKY19 was digested with Pstl and an 8-kb fragment containing the entire phr deletion was used to transform JC7623 (recBC sbcB), pKY34 and pKY36 were digested with Accl and a 4.2-kb fragment for pKY34 and a 3.8-kb fragment for pKY36 containing the phr insertion or deletion were used to transform JC7623. DNA of bacterial origin is indicated by open double lines. Restriction sites: E, EcoRI; P, Pstl; A, Accl; M, Mlul. M/H denotes ligation between blunted Mlul and blunted Haell where the Mlul site generates. (B) Chromosomal DNA blot of phr +, phr-19, phr-34 and phr-36 strains. A total of 2.5 /zg of DNA was loaded in each lane. The DNA blot was probed with a 1.3-kb Mlul-Mlul fragment of pKY33. Lanes 1 and 5, KY116: 2 and 6, KY196(phr-19); 3 and 7, KY197(phr-34); 4 and 8, KY198(phr-36). The DNA was digested with Pstl (lanes 1-4) or EcoRI and Pstl (lanes 5 8). Base pairs (in thousands) are indicated on the right and left of the lanes. c o m p a r e d in a uvrA6 b a c k g r o u n d . Strain K Y l l 6 (uvrA6 phr +) a n d the defective derivatives KY136 (phr-2), KY196 (phr-19), KY197 (phr-34) a n d
31
ooi
0.0%
~
lb
UV(J/m 2)
Fig. 2. The surviving fraction after 254 nm UV and after 2 h illumination of photoreactivating light. Overnight cultures in LB medium were centrifuged, resuspended in buffer and exposed to 254 nm UV light. After the indicated exposures, samples were exposed to continuous photoreactivatinglight for 0 h (open symbols) or 2 h (closed symbols) (daylight fluorescent lamp filtered through polyvinyl chloride plastic which cut out wavelengths below 380 nm). Appropriate dilutions were plated to determine survival. KYll6 (uvrA6 phr ÷) (o, @); KY136(uvrA6 phr-2) (zx, A); KY196(uvrA6 phr-19) (D, II); KY197(uvrA6 phr-34) (0, @); KY198( uvrA6phr-36) (v, v).
KY198 (phr-36) were equally sensitive to UV radiation (Fig. 2). All the phr replacement mutants we constructed and phr-2 (isolated by H a r m and Hillebrandt, 1962) showed no photoreversal of UV killing in 2-h illumination with the fluorescent lamp (Fig. 2). Probably filtering the photoreactivating light with polyvinyl chloride plastic to absorb wavelengths below 380 nm eliminated residual photoreactivation (Husain and Sancer, 1987; Smith et al., 1987). In other experiments, we photoreactivated these strains for up to 6 h. Again we did not detect any photoreactivation in the phr-deficient strain (data not shown). Previously we have shown that an excision-deficient strain with increased D N A photolyase (due to the presence of a multicopy plasmid carrying phr) was more sensitive to inactivation by UV (Yamamoto et al., 1984). This sensitivity was not observed when there was only one copy of phr ÷, since there was no significant difference between KY 116 (uvrA 6 phr ÷) and any of the other isogenic
phr-deficient strains assayed in the dark (Fig. 2, open symbols). We also demonstrated that an excision-proficient recA strain with a multicopy phr plasmid was more UV-resistant than the same strain containing normal amounts of photolyase ( Y a m a m o t o et al., 1983b). We therefore compared the UV sensitivity in the dark of isogenic recA strains with or without the phr-36 defective allele (Fig. 3A) and lexA1 strains with phr ÷ or phr-36 (Fig. 3B). In both cases, survival was significantly greater for the phr ÷ strain than for the phr-36 strain. The results with the recA strain are consistent with our previous work ( Y a m a m o t o et al., 1983a). However, we showed that UV sensitivities of a lexA 1 strain with or without the phr plasmid were almost identical ( Y a m a m o t o et al., 1984). Therefore, we re-examined the effect of the multicopy phr plasmid on the dark survival of a lexA 1 strain and found that the plasmid decreased the dark survival of the lexA1 strain (data not shown). We have no interpretation for the surprising result that 1-copy phr increased and multicopy phr decreased the dark survival of a lexA1 strain. As a whole, in the recA or lexA background, the phr ÷ strain is more resistant than the p h r - strain sug-
~rn
.->
(8)
.
~ 10
TM
\ lO o
\
UV (Jim 2)
Fig. 3. The surviving fraction after 254 nm UV light in E. coli strains carrying mutations in either recA56 or lexAl with phr deficiency. (A) Survival of recA56 with phr deficiency. KYlO56(recA56 phr +) (©); KY1225(recA56 phr-36) (z~). (B) Survival of lexA1 with phr deficiency. KY1071(IexA1 phr + ) ([3); KY1221( lexAl phr-36) (v).
32
gesting that a normal level of DNA photolyase enhances excision-mediated dark repair.
10~
Effect of phr alleles on UV mutagenesis We have shown previously that mutations responsible for His + prototropic mutants were more efficiently produced in strain ABl157 than in the strain with amplified DNA photolyase (Yamamoto et al., 1984). This phenomenon was later interpreted to mean that D N A photolyase bound to the targeting pyrimidine dimer interrupted the mutational DNA synthesis (Ruiz-Rubio et al., 1988). In order to find out whether physiological levels of D N A photolyase still disturb the mutational D N A synthesis at the targeting pyrimidine dimer, we tested the mutability in uvrA6 derivatives with the phr-19 or phr-36 replacement allele. As shown in Fig. 4, phr-deficient strain KY1839(phr-19) and KY1226(phr-36) were consistently more mutable, depending somewhat on the UV dose employed. Pyrimidine dimers produced in DNA would increase proportionally with increasing doses of UV radiation. Therefore, in KY1836(uvrA6 phr ÷), at smaller doses a greater portion of pyrimidine dimers would be bound with DNA photolyase to interrupt mutational D N A synthesis and relatively fewer mutations would be produced. At higher UV doses, more of the targeting pyrimidine di-
[5"
u~ 10.6
-7
7o-8 o:s. ,.. I
~],,~
........ 1
2 3
UV(Jlm 2 )
Fig. 4. Induction of His + mutations after 254 nm UV radiation. Overnight cultures in LB medium were centrifuged, resuspended in buffer and exposed to UV light. After the indicated exposures, samples were plated on each of duplicate SEM places to select for his-4 to His + mutation. After appropriate dilutions, samples were plated on LB agar to count the surviving fraction. Experiments were carried out 3 times and were highly reproducible. Two of the 3 experiments are given to indicate the reproducibility of the results. KY1836( uvrA6 phr ÷) ( o ) ; KY1226(uorA6 phr-36) (z~); KY1839(uvrA6 phr-19) (0).
mers would be free of D N A photolyase even in KY1836(phr +) resulting in more similar mutability compared to KY1839 or KY1226.
TABLE 2 T Y P I C A L Y I E L D S OF SUPPRESSOR M U T A T I O N S BY 1 J / m 2 UV R A D I A T I O N IN uvrA6 D E R I V A T I V E S W I T H phr MUTATIONS a Nam e
N u m b e r of suppressor mutations
Total His ÷ examined
Suppressor mutation frequency b (%)
Mutation frequency ~
KY1836(phr +)
expt. 1 2 3
25 28 27
83 87 71
30 32 35
5.36x10 v 5.83 x 10 7 4.57x10 7
KY1226(phr-36)
expt. 1 2 3
59 63 59
113 115 109
52 55 54
1.13x 10 6 1 . 3 8 x 1 0 -6 1.31 x 10 6
KY1326(phr-34)
expt. 1
59
120
49
9.32 x 10 7
KY1839(phr-19)
expt. 1
49
94
52
2.13X10
6
a Three independent clones for KY1836 and KY1226, and 1 representative clone for KY1326 and KY1839 were incubated overnight in Luria broth. After 1 J / m z UV irradiation, 1-1.5 x 108 cells were placed on SEM agar plates to select for His + prototropy. All the independent His + colonies were picked up from each SEM agar plate and patched on minimal salt agar plates without arginine or histidine to distinguish suppressor mutants and back mutants. b 100 X number of suppressor m u t a t i o n s / t o t a l His + colonies examined. c His + p r o t o t r o p h s / s u r v i v i n g cells.
33 His + mutants can be tested with streaking on minimal medium to determine whether they result from ochre suppressor mutations or back mutations. As shown in Table 2, when the his-4 allele was in KY1836(uvrA6 phr+), 25-34% of His ÷ mutants produced by 1 J / m 2 UV mutagenesis were suppressor mutations. When the his-4 allele was in phr-deficient strains, 50-55% of His + mutants were suppressor mutations.
Discussion This paper describes the construction and characterization of replacement mutation in phr, the gene for DNA photolyase in E. coli. Our procedure was to insert a 1.4-kb cat gene which codes for chloramphenicol acetyltransferase into a phr gene carried on plasmid pKY1 or pKY33. We have been able to obtain 3 well-defined mutations in phr. These mutations were subsequently crossed into the chromosome, replacing the functional phr gene. The resulting mutations had totally lost photoreactibility. The new alleles, including 2 deletions and 1 insertion, were named phr-19 (complete deletion), phr-36 (partial deletion) and phr34 (insertion). The Cm r mutations crossed into the chromosome were 100% linked to phr by P1 transduction and were 10% linked to gal as previously shown for phr (Youngs and Smith, 1978). The uvrA6 strain with these phr alleles and phr-2 were similarly insensitive to photoreactivating light (Fig. 2). We also obtained the results that the recA56 uvrA6 strains with these phr alleles as well as phr-1 (i.e., CSR603) showed no photoreactivation at all (data not shown). These alleles thus showed the null phenotype for photoreactivation. In the absence of photoreactivating light, we demonstrated that recA phr-2 was more sensitive to UV radiation than the recA phr ÷ strain (Yamamoto et al., 1983a). We demonstrated (Fig. 3) that recA56 or lexA1 mutations with phr-36 were more sensitive to UV radiation than recA56 phr ÷ or lexA1 phr ÷. DNA photolyase in the dark is known to enhance nucleotide excision repair (Yamamoto et al., 1983b; Sancer et al., 1984a). Thus, the results indicate that a physiological amount of D N A photolyase has the ability to
manifest the dark role of photolyase in recA and
lexA strains. On the other hand, there was no significant difference in UV sensitivity between uvrA6 phr ÷ and uvrA6phr- (Fig. 2, open symbols). The uvrA6 strain with excess amounts of photolyase due to the presence of the multicopy plasmid was more sensitive to UV radiation than the strain with 1 copy of the phr gene (Yamamoto et al., 1984). The results suggest that the sensitization mediated by photolyase in excision-deficient strains can be seen only when there are high amounts of D N A photolyase. It has recently been reported that E. coli K12 strains mutated in the phr gene have residual photoreactivation activity (Husain and Sancer, 1987; Smith et al., 1987). The possibility that this residual activity may be due to the direct photoreversal of (6-4)photoproducts was proposed (Husain et al., 1988). In our photoreactivation condition, in which photoreactivating light delivered from a daylight fluorescent lamp was filtered through a 0.2-cm layer of polyvinyl chloride plastic to cut out wavelengths below 380 nm, we have not observed any residual photoreactivation for up to 2 h continuous illumination in newly isolated phr mutations and previously isolated phr-2 (Fig. 2, closed symbols). We have also not detected any residual photoreactivation in KY196(uvrA6 phr19), KY197( uvrA6 phr-34) and KY198( uvrA6 phr36) after illumination for up to 6 h (data not shown). If the so-called residual photoreactivation is due to direct photoconversion of the (6-4)photoproducts as suggested by Husain et al. (1988), the effective wavelength for the phenomenon might be 300-360 nm with a peak at 313 nm (Ikenaga and Jagar, 1971). It is therefore possible that we did not observe any residual photoreactivation because we used polyvinyl chloride plastic to remove the effective wavelength. Our data indicate that the sequences flanking phr for 300 bp upstream and 4 kb downstream of the gene are not essential for E. coli growth. As shown by D N A sequence analysis (Sancer et al., 1984b), immediately preceding the phr is an open reading frame of orf169 encoding a 20-kDa protein. Orf169 was deleted in phr-19 and phr-36. There was no significant difference in UV sensitivity or UV mutability between these 2 mutants and
34
the phr + strain or phr-34 (both are orf169+), For example, as far as orf169 is concerned, UV sensitivities as shown in Fig. 2 (open symbols) were equal in these strains. UV mutabilities in strains with phr-19, phr-34 and phr-36 were again equal (data not shown) and the rates of suppressor mutations produced by UV in these strains were the same (Table 2). Thus, the orf169 gene product is not essential for growth nor has it a direct or regulating role for D N A repair. When we c o m p a r e d UV mutagenesis in KY1836(uorA6 phr +) and the phr-deficient derivatives by measuring the induction of His + prototropic mutants, substantially more mutants resulted with the phr-deficient strains (Fig. 4). Individual mutant colonies were picked up and tested to identify His + mutants as back or suppressor mutants. The increased mutation frequency response in cells with phr deficiency was found to be due to an increase of the suppressor mutations (Table 2). Suppressor mutations are specific basepair substitution in t R N A genes (Ozeki et al., 1980), with the most frequent of these at genes for glutamine tRNA. The glutamine t R N A ochre suppressor mutations, de novo and converted, respectively, require G C to AT transitions at either end of the anticodons: -ATCAAAA- and - A T T A G A A (transcribed strands ~or the anticodon lo--ops). These are well characterized and efficiently produced by conventional UV mutagenesis (Bockrath et al., 1987; Engstrom et al., 1984; Kato et al., 1980). There are several facts indicating that glutamine t R N A suppressor mutations can be targeted at T = C pyrimidine dimers after UV irradiation (Bockrath et al., 1987). Thus, in uvrA6 strains with phr deficiency, there is a specific increase of mutations associated with targeted T
possesses higher amounts of photolyase per cell than a strain with a moderate mutation frequency, and the strain with the highest mutation frequency is defective for phr. These data support the earlier suggestion that D N A photolyase interferes with UV mutagenesis by virtue of binding to the targeting T = C dimer to interrupt mutational D N A synthesis. Recently evidence has been obtained that, in E. coli, D N A photolyase plays varying roles in the dark in the D N A metabolism: stimulation of excision repair in the recA strain, sensitization of excision-deficient strains and reduction of mutations associated with targeting by T = C. Such a dark role of D N A photolyase is not specific for E. coli. Sancer and Smith (1989) have reported that phr + strains of Saccharomyces cerevisiae are more resistant to UV killing than phr-deficient strains in a radl8 background. A photorepair-deficient mutant in Drosophila melanogaster was found to be partially deficient in dark repair and the deficiency was closely linked to a phr mutation (Boyd and Harris, 1987). Thus, in general, in addition to its role in light-dependent repair, D N A photolyase has a role in light-independent repair. As a consequence, if one wants to consider specific repair activities that alter the effect of UV radiation, the use of strains with phr deficiencies can reduce the complexity of interacting effects due to the formation of D N A photolyase and pyrimidine dimer complexes. The alleles we constructed are useful for such a purpose, since the alleles can easily be transferred to the strain of interest just by selecting drug resistance.
~C.
We thank Dr. B.J. Bachmann for providing the strains and Dr. R. Bockrath for critically reading the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Area ' T h e Molecular Mechanism of Photoreception' from the Ministery of Education, Science and Culture of Japan.
It has been demonstrated previously that in strains carrying a large amount of D N A photolyase due to the presence of a multicopy plasmid carrying phr, production of glutamine t R N A suppressor mutations by UV radiation was reduced compared to strains with normal amounts of photolyase (Ruiz-Rubio et al., 1988). Therefore, there is a very good inverse correlation between the frequency of mutations associated with targeting T = C and the amounts of D N A photolyase per cell; the strain with the lowest mutation frequency
Acknowledgements
References Bockrath, R., M. Ruiz-Rubio and B.A. Bridges (1987) Specificity of mutation by UV light and delayed photoreversal in
35 umuC-defective Escherichia coli K-12: a targeting intermediate at pyrimidine dimers, J. Bacteriol., 169, 1410-1416. Boyd, J.B., and P.V. Harris (1987) Isolation and characterization of a photorepair-deficient mutant in Drosophila melanogaster, Genetics, 116, 233-239. Engstrom, J., S. Larsen, S. Rogers and R. Bockrath (1984) UV mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation, Mutation Res., 132, 143-152. Harm, W., and B. Hillebrandt (1962) A nonphotoreactivable mutant of E. coli B, Photochem. Photobiol., 1,271-272. Hays, J.B., S.J. Martin and K. Bhatis (1985) Repair of non replicating UV-irradiated DNA: cooperative dark repair by Escherichia coli Uvr and Phr functions, J. Bacteriol., 161, 602-608. Howard-Flanders, P., and L. Theriot (1965) Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination, Genetics, 53, 1137-1150. Husain, I., and A. Sancer (1987) Photoreactivation in phr mutants of Escherichia coli K-12, J. Bacteriol., 169, 23672372. Husain, I., W.L. Carrier, J.D. Regan and A. Sancer (1988) Photoreactivation of killing in E. coli K-12 phr- cells is not caused by pyrimidine dimer reversal, Photochem. Photobiol., 48, 233-234. Ihara, M., Y. Oda and K. Yamamoto (1985) Convenient construction of strains useful to transducing recA mutations with bacteriophage P1, FEMS Microbiol. Lett., 30, 33-35. Ikenaga, M., and J. Jagar (1971) Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus phr-l, Photochem. Photobiol., 13, 459-471. Kato, T., S. Shinoura, A. Templin and A.J. Clark (1980) Analysis of ultraviolet light-induced suppressor mutations in the strain Escherichia coli K-12 ABl157: an implication for molecular mechanisms of UV mutagenesis, Mol. Gen. Genet., 180, 283-291. Kushner, S., H. Nagaishi, A. Templin and A.J. Clark (1971) Genetic recombination in Escherichia coli: the role of exonuclease I, Proc. Natl. Acad. Sci. (U.S.A.), 68, 824-827. Ozeki, H., H. Inokuchi, F. Yamao, M. Kodaira, H. Sakano, T. Ikemura and Y. Shimura (1980) Genetics of nonsense suppressor tRNA in Escherichia coli, in: D. S~II, J.N. Abelson and P.R. Schimmel (Eds.), Transfer RNA: Biological Aspects, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 341-362. Rhoads, D.B., L. Laimins and W. Epstein (1978) Functional organization of the kdp genes of Escherichia coli K-12, J. Bacteriol., 135, 445-452. Ruiz-Rubio, M., K. Yamamoto and R. Bockrath (1988) An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli, J. Bacterioi., 170, 5371-5374.
Rupert, C.S. (1975) Enzymatic photoreactivation: overview, in P.C. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Part A, Plenum Press, New York, pp. 73-87. Sancer, A., and C.S. Rupert (1978a) Correlation of the map location for the phr gene in Escherichia coli K-12, Mutation Res., 51, 139-143. Sancer, A., and C.S. Rupert (1978b) Determination of plasmid molecular weights from ultraviolet sensitivities, Nature (London), 272, 471-472. Sancer, G.B., and F.W. Smith (1989) Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and E. coli, Mol. Cell. Biol., 9, 4767-4776. Sancer, A., K.A. Franklin and G.B. Sancer (1984a) Escherichia coli DNA photolyase stimulates UvrABC excision nuclease in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 81, 7397-7401. Sancer, G.B., F.W. Smith, M.C. Lorence, C.S. Rupert and A. Sancer (1984b) Sequence of the 15. coli photolyase gene and p~:otein, J. Biol. Chem., 259, 6033-6038. Smith, A.W., J.G. Davies and S.H. Moss (1987) Photoreactivation of ultraviolet radiation damage in dark repair deficient phr mutants of Escherichia coli K-12, Photochem. Photobiol., 45, 247-252. Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis, J. Mol. Biol., 98, 503-517. Walker, G.C. (1984) Mutagenesis and inducible response to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Winans, S.C., S.J. Elledge, J.H. Krueger and G.C. Walker (1985) Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli, J. Bacteriol., 161, 1219-1221. Yamamoto, K., and R. Bockrath (1989) DNA photolyase in E. coli: effects on UV mutagenesis by plasmids expressing the phr gene, Mutation Res., 226, 259-262. Yamamoto, K., Y. Fujiwara and H. Shinagawa (1983a) Evidence that the phr + gene enhances the ultraviolet resistance of Escherichia coli recA strains in the dark, Mol. Gen. Genet., 192, 282-284. Yamamoto, K., M. Satake, H. Shinagawa and Y. Fujiwara (1983b) Amelioration of the ultraviolet sensitivity of an Escherichia coli recA mutant in the dark by photoreactivating enzyme, Mol. Gen. Genet., 190, 511-515. Yamamoto, K., M. Sataka and H. Shinagawa (1984) A multicopy phr plasmid increases ultraviolet resistance of a recA strain of Escherichia coli, Mutation Res., 131, 11-18. Youngs, D.A., and K.C. Smith (1978) Genetic location of the phr gene of Escherichia coli K-12, Mutation Res., 51, 131-137.
27
MUTDNA 06404
Construction of Escherichia coli K12 phr deletion and insertion mutants by gene replacement Susumu Akasaka i and Kazuo Yamamoto 2 +Division of Industrial Health, Osaka Prefectural Institute of Public Health, Higashinari, Osaka 537 and 2 Biological Institute, Faculty of Science, Tohoku University, Sendai 980 (Japan)
(Received 16 January 1990) (Revision received 11 May 1990) (Accepted 16 May 1990)
Keywords: phr replacement mutants; DNA photolyase; Pyrimidine dimers; Suppressor mutations
Summary We replaced an Escherichia coli phr gene by a 1.4-kb fragment of DNA coding for resistance to chloramphenicol. Characterization of 2 deletions (phr-19 and phr-36) and 1 insertion (phr-34) in the phr gene revealed no photoreactivation. Photoreactivation-deficient strains of either recA56 or lexAl(ind-) were more sensitive to UV radiation in the dark than phr-proficient counterparts. The presence of the phr defect in uvrA6 strains increased by 1.5-2-fold his-4(Ochre) to His + mutation induced by ultraviolet light compared to uvrA6 phr ÷ strains, although there was no difference in UV sensitivity between uvrA6 phr ÷ and uvrA6 phr- strains. 30-35% of the His ÷ mutations thus induced were suppressor mutations in uvrA6 phr ÷ and 49-55% in uvrA6 phr- strains. The UV mutagenesis results are consistent with the previous observations that suppressor mutations targeted by a thymine-cytosine pyrimidine dimer are reduced in the dark in cells with amplified DNA photolyase.
Cis-syn cyclobutane dipyrimidine dimers (pyrimidine dimers) are induced in DNA by 254 nm ultraviolet light and are one of the primary lesions responsible for UV-induced carcinogenesis, mutation and cell lethality. Enzymatic photoreactivation, mediated by DNA photolyase which is coded by the phr gene at 16.2 min on the Escherichia coli linkage map (Sancer and Rupert, 1978a; Youngs and Smith, 1978), is one pathway by which these lesions are repaired; pyrimidine dimers are bound by photolyase which subsequently absorbs a pho-
Correspondence: K. Yamamoto, Biology Institute, Faculty of Science, Tohoku University, Sendal 980 (Japan).
ton of near-UV light and utilizes this energy to cleave the cyclobutane ring linking the 2 pyrimidines (Rupert, 1975). In addition to photoreactivation, E. coli possesses other repair pathways by which pyrimidine dimers are removed from DNA or genotoxic effects of these lesions are bypassed. These include excision repair, recombination repair and SOS repair (Walker, 1984). Because pyrimidine dimers in DNA are potential substrates for several repair pathways in the same cell, interactions between components of these pathways must be considered in assessing the contribution of each pathway to repair and damage tolerance. Such a notion was supported by a number of recent studies which
0921-8777/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
28
have demonstrated that, even in the absence of photoreactivating light, bound photolyase plays a repairing role in E. coli in the dark. E. coli recA phr ÷ strains are more resistant to UV-induced cell killing than are recA phr- strains (Yamamoto et al., 1983a; Hays et al., 1984). E. coli photolyaseoverproducing uvr-deficient strains are more UVsensitive than uvr-deficient strains producing normal enzyme levels (Yamamoto et al., 1984). With cells containing amplified amounts of photolyase, mutations targeted at thymine-cytosine pyrimidine dimers (T = C) are greatly reduced or eliminated compared to cells containing normal levels of photolyase (Ruiz-Rubio et al., 1988). The fact that photolyase can bind to pyrimidine dimers and then manifest repair effects in the absence of light relates to the question of how much each of the repair pathways contributes to the observed UV results. A genetic approach to answer this question would use isogenic pairs of phr ÷ and phr- strains and compare the effects of UV radiation. There are phr mutants available: phr-1 (Sancer and Rupert, 1978b), phr-2 (Harm
and Hillebrandt, 1962), A(kdp-gltA)214 (Hays et al., 1984) and A(kdp-gltA)219 (Rhoads et al., 1978). However, since a good selective marker for phr does not exist, P1 phage transduction using these alleles has sometimes encountered difficulty. In this paper we describe how we inserted a selectable marker, resistance to chloramphenicol ( C m r ) , into a phr gene and introduced it into the bacterial chromosome by homologous recombination (Winans et al., 1985). The new alleles can easily be transduced by the P1 phage to construct isogenic pairs of phr ÷ and phr- strains. Analysis of the resulting strains suggests that (i) the photoreactivation-defective phenotypes of these mutants are similar to those of mutants with previously isolated phr-1 and phr-2 genes, (ii) a recA phr + strain is more resistant to UV than a recA phr strain in the dark, (iii) a lexA(ind- ) phr + strain is more UV-resistant than a lexA(ind-) phr strain in the dark, (iv) there are no significant differences in UV sensitivity between uvrA phr ÷ and uvrA phr strains in the dark and (v) uvrA phr- strains are more mutable than uvrA phr + strains. UV
TABLE 1 LIST OF STRAINS Name
Relevant genotype
Reference or source
argE3 his-4 leu-6 proA2 thr-I thi-1 rpsL31 galK2 lacY1 ara-14 xyl-5 mtl-1 supE44 As ABl157 but uvrA6 As KY1836 but phr-36 As KY1836 but phr-19 As KY1836 but phr-34 As ABl157 but phr-36 As ABl157 but srlC300:: TnlO recA56 As KY1220 but srlC300: : TnlO recA56 As ABl157 but malE :: TnlO lexA1 As KY1220 but malE :: TnlO lexA1 purA45 uvrA6 gal-6 As K Y l l 6 but phr-2 gal + As K Y l l 6 but phr-19 As K Y l l 6 but phr-34 As K Y l l 6 but phr-36 recB21 recC22 sbcBl5
Howard-Flanders and Theriot (1965) This work This work This work This work This work Yamamoto et al. (1984) This work This work This work Yamamoto et al. (1983b) Yamamoto et al. (1983b) This work This work This work Kushner et al. (1971)
pBR322 + 20-kb phr fragment pBR322 + 2.8-kb phr fragment As pKY1 but phr:: cat ( = phr-19) As pKY33 but phr :: cat ( = phr-34) As pKY33 but phr:: cat ( = phr-36)
Yamamoto et al. (1983a) Ruiz-Rubio et al. (1988) This work Yamamoto and Bockrath (1989) Yamamoto and Bockrath (1989)
E. coli ABl157 KY1836 KY1226 KY1839 KY1326 KY1220 KY1056 KY1225 KY 1701 KYI221 KYll6 KY136 KY196 KY197 KY 198 JC7623
Plasmid pKY1 pKY33 pKY19 pKY34 pKY36
29 mutagenesis targeted at T = C increases in a uvrA phr- strain compared to a uvrA phr + strain.
growth were carried out under yellow light to prevent uncontrolled photoreactivation.
Materials and methods
Survival and mutation assays After UV irradiation, and photoreactivation if applied, surviving colonies were counted following overnight incubation at 37 ° C on Luria agar plates. His ÷ prototrophic mutants were counted, and the samples were isolated after the plates had been incubated for 3 days at 37 ° C. Mutation frequencies were calculated by subtracting the numbers of spontaneous colonies per plate from the numbers of colonies on plates inoculated with treated cells and then dividing these net numbers by the numbers of viable cells assayed per plate. Since both his-4 and argE3 markers in derivatives of strain ABl157 are ochre nonsense mutations and suppressed by glutamine tRNA ochre suppressor mutations (Kato et al., 1980), one can easily identify His + prototrophic mutants as being back or suppressor mutants. All His ÷ mutants per plate (approximately 100 colonies) were patched on minimal salt agar minus arginine and minus histidine plates. Suppressor mutants were identified as those that were able to demonstrate growth on both plates. Back mutants were those that could grow only on the histidine-free plates.
Bacterial strains and plasmids The bacterial strains and plasmids used in this work are listed in Table 1. P1 phage transduction was used to construct isogenic pairs of strains (Ihara et al., 1985). The constructions of plasmids utilized in this work except pKY19 have been reported previously (Yamamoto et al., 1983b; Yamamoto and Bockrath, 1989). Growth media Cultures to be irradiated were grown in Luria broth. Phosphate buffer (4.5 g KH2PO 4, 4.7 g N a 2 H P O 4 per liter of distilled water, p H 6.8) was used for washing, resuspending and diluting cells. Minimal salt agar medium was made of M56 salt plus 0.35% glucose, 1.5% agar supplemented with thiamine (1 /~g/ml) and the amino acids proline, threonine, leucine, histidine and arginine (each at 100 /Lg/ml). His + prototropic mutants were selected on semi-enriched minimal agar (SEM, minimal salt agar medium plus 0.004% casamino acids and no histidine). Minimal salt agar medium lacking histidine or arginine plates was used for His + mutant differentiation. UV radiation and photoreactivation Overnight, stationary-phase cultures of E. coli strains were washed in phosphate buffer, and resuspended in the buffer at about 5 × 108/ml. Cells in 90 mm diameter glass petri dishes were exposed to 254 nm UV light with constant shaking using 2 15-W germicidal lamps (Matsushita Electric Co.). The fluence rate measured by a Topcon UV dosimeter was 0.66 j / m 2 / s . For photoreactivation, 2 Matsushita daylight fluorescent lamps at 20 cm distance were use as the light source. Polyvinyl chloride plastic (0.2 cm layer) and a glass petri dish lid were placed between the photoreactivation light and the sample aliquots in buffer. That the polyvinyl chloride plastic used in this experiment absorbs wavelengths below 380 nm was established with an ISCO UVIDEC 610E spectrophotometer. All irradiation, subsequent manipulations and
Southern blotting The chromosomal D N A blot was probed with a 1.3-kb MluI-MluI phr fragment of pKY33 which was terminal-labeled with [y-32p]ATP with T4 polynucleotide kinase (Takara). Hybridization was done by the procedure of Southern (1975). Results
Construction of the phr deletion and insertion mutations Plasmid pKY1 and the derivative pKY33 carrying the phr gene were used to receive insertions of a chloramphenicol acetyltransferase (cat) gene derived from pACYC184. Plasmid pKY33 contains a 2.8-kb AccI fragment from pKY1, which in turn carries a 20-kb EcoRI fragment from the E. coli chromosome that includes the phr gene. There are 3 MluI sites in pKY33 and 4 in pKY1. The recessed termini of linear isolates from pKY1 after full digestion with MluI or from pKY33 after
3o partial digestion with Mlul were filled in with K l e n o w polymerization, a n d these were ligated to a 1.4-kb HaelI f r a g m e n t from p A C Y C 1 8 4 (cont a i n i n g cat) that was digested to b l u n t ends with Klenow. Restriction maps were then d e t e r m i n e d of several isolated plasmids that conferred both ampicillin a n d c h l o r a m p h e n i c o l resistance a n d were phr-deficient. This i d e n t i f i e d p l a s m i d s p K Y 1 9 , p K Y 3 4 a n d p K Y 3 6 (Fig. 1A). Plasmid p K Y 1 9 was derived from pKY1. A n 8-kb PstI linear fragment carrying the cat gene within a deletion from the first to the fourth Mlul site, that is 300 b p u p s t r e a m a n d 4 kb d o w n s t r e a m of the phr, could be isolated from pKY19. Plasmids p K Y 3 4 a n d p K Y 3 6 were derived from pKY33. A 4.2-kb AccI linear fragment from p K Y 3 4 a n d a 3.9-kb fragment from p K Y 3 6 carrying the cat insertions could be isolated from p K Y 3 4 a n d pKY36, respectively. The former contained the insertion after the first 88 bp of the p r o t e i n coding sequence of phr. The latter had the cat insertion in place of a deletion between the p r o m o t e r area (300 bp u p s t r e a m of the phr) a n d the 88th bp of the phr sequence. The linear fragments thus isolated c o n t a i n i n g cat insertions were used to transform a c o m p e t e n t recB21 recC22 sbcB15 strain (JC7623) ( K u s h n e r et al., 1971). T r a n s f o r m a n t s were selected for C m r. P1 lysates were m a d e on the J C 7 6 2 3 : : cat transf o r m a n t s a n d were used to transduce a p p r o p r i a t e strains to C m r. The C m r p h e n o t y p e proved to be a p p r o x i m a t e l y 10% c o t r a n s d u c i b l e with gal by P1 t r a n s d u c t i o n , as was expected for the phr locus ( Y o u n g s a n d Smith, 1978). C m r t r a n s d u c t a n t s of a uurA6 phr + strain ( K Y I 1 6 ) were e x a m i n e d by a streak test for phr + or phr a n d were all f o u n d to be phr-deficient. C h r o m o s o m a l D N A blots of a phr + strain a n d 3 CmLderived strains c o n f i r m e d that these cells contained the desired phr deletions or insertions (Fig. 1B). W e refer to the phr replacement alleles as phr-19, phr-34 a n d phr-36 (from pKY19, p K Y 3 4 a n d pKY36, respectively).
U V inactivation and photoreactivation of phr replacement mutants T o d e t e r m i n e whether phr replacement m u t a n t s differed from the previously isolated phr m u t a n t s , the UV sensitivity a n d photoreactivability were
(A)
E
P P I
pK¥1 ', pKY33
E
I
\11--/[
I
ANM
M
"I
r
P P ,., i
pKY19 I
AM~NAP M P
M/HE
I
E
I
I
A
' ivl/H P
~
I
E
,
I
pKY34
pKY36
(B)
123
45
678
-23
9-4~ 9.0- .~.
8.0/ #7-
9.6
6-6
4-4 2.3
Fig. 1. Construction of phr deletions and insertion. (A) pKY1 has a 20 kb-fragment containing the phr gene (solid rectangle) inserted at the EcoRl site in pBR322 (Yamamoto et al., 1983b). pKY33 is constructed from pBR322 with insertion of a 2.8-kb Accl fragment containing the phr gene from pKY1 via pBG6 (Ruiz-Rubio et al., 1988). We cloned a blunted 1.4-kb Haell fragment containing the (.at gene (hatched double line), derived from pACYC184, into blunted Mlul sites into pKY33 to generate pKY34 and pKY36 (Yamamoto and Bockrath, 1989), and into pKY1 to generate pKY19, pKY19 was digested with Pstl and an 8-kb fragment containing the entire phr deletion was used to transform JC7623 (recBC sbcB), pKY34 and pKY36 were digested with Accl and a 4.2-kb fragment for pKY34 and a 3.8-kb fragment for pKY36 containing the phr insertion or deletion were used to transform JC7623. DNA of bacterial origin is indicated by open double lines. Restriction sites: E, EcoRI; P, Pstl; A, Accl; M, Mlul. M/H denotes ligation between blunted Mlul and blunted Haell where the Mlul site generates. (B) Chromosomal DNA blot of phr +, phr-19, phr-34 and phr-36 strains. A total of 2.5 /zg of DNA was loaded in each lane. The DNA blot was probed with a 1.3-kb Mlul-Mlul fragment of pKY33. Lanes 1 and 5, KY116: 2 and 6, KY196(phr-19); 3 and 7, KY197(phr-34); 4 and 8, KY198(phr-36). The DNA was digested with Pstl (lanes 1-4) or EcoRI and Pstl (lanes 5 8). Base pairs (in thousands) are indicated on the right and left of the lanes. c o m p a r e d in a uvrA6 b a c k g r o u n d . Strain K Y l l 6 (uvrA6 phr +) a n d the defective derivatives KY136 (phr-2), KY196 (phr-19), KY197 (phr-34) a n d
31
ooi
0.0%
~
lb
UV(J/m 2)
Fig. 2. The surviving fraction after 254 nm UV and after 2 h illumination of photoreactivating light. Overnight cultures in LB medium were centrifuged, resuspended in buffer and exposed to 254 nm UV light. After the indicated exposures, samples were exposed to continuous photoreactivatinglight for 0 h (open symbols) or 2 h (closed symbols) (daylight fluorescent lamp filtered through polyvinyl chloride plastic which cut out wavelengths below 380 nm). Appropriate dilutions were plated to determine survival. KYll6 (uvrA6 phr ÷) (o, @); KY136(uvrA6 phr-2) (zx, A); KY196(uvrA6 phr-19) (D, II); KY197(uvrA6 phr-34) (0, @); KY198( uvrA6phr-36) (v, v).
KY198 (phr-36) were equally sensitive to UV radiation (Fig. 2). All the phr replacement mutants we constructed and phr-2 (isolated by H a r m and Hillebrandt, 1962) showed no photoreversal of UV killing in 2-h illumination with the fluorescent lamp (Fig. 2). Probably filtering the photoreactivating light with polyvinyl chloride plastic to absorb wavelengths below 380 nm eliminated residual photoreactivation (Husain and Sancer, 1987; Smith et al., 1987). In other experiments, we photoreactivated these strains for up to 6 h. Again we did not detect any photoreactivation in the phr-deficient strain (data not shown). Previously we have shown that an excision-deficient strain with increased D N A photolyase (due to the presence of a multicopy plasmid carrying phr) was more sensitive to inactivation by UV (Yamamoto et al., 1984). This sensitivity was not observed when there was only one copy of phr ÷, since there was no significant difference between KY 116 (uvrA 6 phr ÷) and any of the other isogenic
phr-deficient strains assayed in the dark (Fig. 2, open symbols). We also demonstrated that an excision-proficient recA strain with a multicopy phr plasmid was more UV-resistant than the same strain containing normal amounts of photolyase ( Y a m a m o t o et al., 1983b). We therefore compared the UV sensitivity in the dark of isogenic recA strains with or without the phr-36 defective allele (Fig. 3A) and lexA1 strains with phr ÷ or phr-36 (Fig. 3B). In both cases, survival was significantly greater for the phr ÷ strain than for the phr-36 strain. The results with the recA strain are consistent with our previous work ( Y a m a m o t o et al., 1983a). However, we showed that UV sensitivities of a lexA 1 strain with or without the phr plasmid were almost identical ( Y a m a m o t o et al., 1984). Therefore, we re-examined the effect of the multicopy phr plasmid on the dark survival of a lexA 1 strain and found that the plasmid decreased the dark survival of the lexA1 strain (data not shown). We have no interpretation for the surprising result that 1-copy phr increased and multicopy phr decreased the dark survival of a lexA1 strain. As a whole, in the recA or lexA background, the phr ÷ strain is more resistant than the p h r - strain sug-
~rn
.->
(8)
.
~ 10
TM
\ lO o
\
UV (Jim 2)
Fig. 3. The surviving fraction after 254 nm UV light in E. coli strains carrying mutations in either recA56 or lexAl with phr deficiency. (A) Survival of recA56 with phr deficiency. KYlO56(recA56 phr +) (©); KY1225(recA56 phr-36) (z~). (B) Survival of lexA1 with phr deficiency. KY1071(IexA1 phr + ) ([3); KY1221( lexAl phr-36) (v).
32
gesting that a normal level of DNA photolyase enhances excision-mediated dark repair.
10~
Effect of phr alleles on UV mutagenesis We have shown previously that mutations responsible for His + prototropic mutants were more efficiently produced in strain ABl157 than in the strain with amplified DNA photolyase (Yamamoto et al., 1984). This phenomenon was later interpreted to mean that D N A photolyase bound to the targeting pyrimidine dimer interrupted the mutational DNA synthesis (Ruiz-Rubio et al., 1988). In order to find out whether physiological levels of D N A photolyase still disturb the mutational D N A synthesis at the targeting pyrimidine dimer, we tested the mutability in uvrA6 derivatives with the phr-19 or phr-36 replacement allele. As shown in Fig. 4, phr-deficient strain KY1839(phr-19) and KY1226(phr-36) were consistently more mutable, depending somewhat on the UV dose employed. Pyrimidine dimers produced in DNA would increase proportionally with increasing doses of UV radiation. Therefore, in KY1836(uvrA6 phr ÷), at smaller doses a greater portion of pyrimidine dimers would be bound with DNA photolyase to interrupt mutational D N A synthesis and relatively fewer mutations would be produced. At higher UV doses, more of the targeting pyrimidine di-
[5"
u~ 10.6
-7
7o-8 o:s. ,.. I
~],,~
........ 1
2 3
UV(Jlm 2 )
Fig. 4. Induction of His + mutations after 254 nm UV radiation. Overnight cultures in LB medium were centrifuged, resuspended in buffer and exposed to UV light. After the indicated exposures, samples were plated on each of duplicate SEM places to select for his-4 to His + mutation. After appropriate dilutions, samples were plated on LB agar to count the surviving fraction. Experiments were carried out 3 times and were highly reproducible. Two of the 3 experiments are given to indicate the reproducibility of the results. KY1836( uvrA6 phr ÷) ( o ) ; KY1226(uorA6 phr-36) (z~); KY1839(uvrA6 phr-19) (0).
mers would be free of D N A photolyase even in KY1836(phr +) resulting in more similar mutability compared to KY1839 or KY1226.
TABLE 2 T Y P I C A L Y I E L D S OF SUPPRESSOR M U T A T I O N S BY 1 J / m 2 UV R A D I A T I O N IN uvrA6 D E R I V A T I V E S W I T H phr MUTATIONS a Nam e
N u m b e r of suppressor mutations
Total His ÷ examined
Suppressor mutation frequency b (%)
Mutation frequency ~
KY1836(phr +)
expt. 1 2 3
25 28 27
83 87 71
30 32 35
5.36x10 v 5.83 x 10 7 4.57x10 7
KY1226(phr-36)
expt. 1 2 3
59 63 59
113 115 109
52 55 54
1.13x 10 6 1 . 3 8 x 1 0 -6 1.31 x 10 6
KY1326(phr-34)
expt. 1
59
120
49
9.32 x 10 7
KY1839(phr-19)
expt. 1
49
94
52
2.13X10
6
a Three independent clones for KY1836 and KY1226, and 1 representative clone for KY1326 and KY1839 were incubated overnight in Luria broth. After 1 J / m z UV irradiation, 1-1.5 x 108 cells were placed on SEM agar plates to select for His + prototropy. All the independent His + colonies were picked up from each SEM agar plate and patched on minimal salt agar plates without arginine or histidine to distinguish suppressor mutants and back mutants. b 100 X number of suppressor m u t a t i o n s / t o t a l His + colonies examined. c His + p r o t o t r o p h s / s u r v i v i n g cells.
33 His + mutants can be tested with streaking on minimal medium to determine whether they result from ochre suppressor mutations or back mutations. As shown in Table 2, when the his-4 allele was in KY1836(uvrA6 phr+), 25-34% of His ÷ mutants produced by 1 J / m 2 UV mutagenesis were suppressor mutations. When the his-4 allele was in phr-deficient strains, 50-55% of His + mutants were suppressor mutations.
Discussion This paper describes the construction and characterization of replacement mutation in phr, the gene for DNA photolyase in E. coli. Our procedure was to insert a 1.4-kb cat gene which codes for chloramphenicol acetyltransferase into a phr gene carried on plasmid pKY1 or pKY33. We have been able to obtain 3 well-defined mutations in phr. These mutations were subsequently crossed into the chromosome, replacing the functional phr gene. The resulting mutations had totally lost photoreactibility. The new alleles, including 2 deletions and 1 insertion, were named phr-19 (complete deletion), phr-36 (partial deletion) and phr34 (insertion). The Cm r mutations crossed into the chromosome were 100% linked to phr by P1 transduction and were 10% linked to gal as previously shown for phr (Youngs and Smith, 1978). The uvrA6 strain with these phr alleles and phr-2 were similarly insensitive to photoreactivating light (Fig. 2). We also obtained the results that the recA56 uvrA6 strains with these phr alleles as well as phr-1 (i.e., CSR603) showed no photoreactivation at all (data not shown). These alleles thus showed the null phenotype for photoreactivation. In the absence of photoreactivating light, we demonstrated that recA phr-2 was more sensitive to UV radiation than the recA phr ÷ strain (Yamamoto et al., 1983a). We demonstrated (Fig. 3) that recA56 or lexA1 mutations with phr-36 were more sensitive to UV radiation than recA56 phr ÷ or lexA1 phr ÷. DNA photolyase in the dark is known to enhance nucleotide excision repair (Yamamoto et al., 1983b; Sancer et al., 1984a). Thus, the results indicate that a physiological amount of D N A photolyase has the ability to
manifest the dark role of photolyase in recA and
lexA strains. On the other hand, there was no significant difference in UV sensitivity between uvrA6 phr ÷ and uvrA6phr- (Fig. 2, open symbols). The uvrA6 strain with excess amounts of photolyase due to the presence of the multicopy plasmid was more sensitive to UV radiation than the strain with 1 copy of the phr gene (Yamamoto et al., 1984). The results suggest that the sensitization mediated by photolyase in excision-deficient strains can be seen only when there are high amounts of D N A photolyase. It has recently been reported that E. coli K12 strains mutated in the phr gene have residual photoreactivation activity (Husain and Sancer, 1987; Smith et al., 1987). The possibility that this residual activity may be due to the direct photoreversal of (6-4)photoproducts was proposed (Husain et al., 1988). In our photoreactivation condition, in which photoreactivating light delivered from a daylight fluorescent lamp was filtered through a 0.2-cm layer of polyvinyl chloride plastic to cut out wavelengths below 380 nm, we have not observed any residual photoreactivation for up to 2 h continuous illumination in newly isolated phr mutations and previously isolated phr-2 (Fig. 2, closed symbols). We have also not detected any residual photoreactivation in KY196(uvrA6 phr19), KY197( uvrA6 phr-34) and KY198( uvrA6 phr36) after illumination for up to 6 h (data not shown). If the so-called residual photoreactivation is due to direct photoconversion of the (6-4)photoproducts as suggested by Husain et al. (1988), the effective wavelength for the phenomenon might be 300-360 nm with a peak at 313 nm (Ikenaga and Jagar, 1971). It is therefore possible that we did not observe any residual photoreactivation because we used polyvinyl chloride plastic to remove the effective wavelength. Our data indicate that the sequences flanking phr for 300 bp upstream and 4 kb downstream of the gene are not essential for E. coli growth. As shown by D N A sequence analysis (Sancer et al., 1984b), immediately preceding the phr is an open reading frame of orf169 encoding a 20-kDa protein. Orf169 was deleted in phr-19 and phr-36. There was no significant difference in UV sensitivity or UV mutability between these 2 mutants and
34
the phr + strain or phr-34 (both are orf169+), For example, as far as orf169 is concerned, UV sensitivities as shown in Fig. 2 (open symbols) were equal in these strains. UV mutabilities in strains with phr-19, phr-34 and phr-36 were again equal (data not shown) and the rates of suppressor mutations produced by UV in these strains were the same (Table 2). Thus, the orf169 gene product is not essential for growth nor has it a direct or regulating role for D N A repair. When we c o m p a r e d UV mutagenesis in KY1836(uorA6 phr +) and the phr-deficient derivatives by measuring the induction of His + prototropic mutants, substantially more mutants resulted with the phr-deficient strains (Fig. 4). Individual mutant colonies were picked up and tested to identify His + mutants as back or suppressor mutants. The increased mutation frequency response in cells with phr deficiency was found to be due to an increase of the suppressor mutations (Table 2). Suppressor mutations are specific basepair substitution in t R N A genes (Ozeki et al., 1980), with the most frequent of these at genes for glutamine tRNA. The glutamine t R N A ochre suppressor mutations, de novo and converted, respectively, require G C to AT transitions at either end of the anticodons: -ATCAAAA- and - A T T A G A A (transcribed strands ~or the anticodon lo--ops). These are well characterized and efficiently produced by conventional UV mutagenesis (Bockrath et al., 1987; Engstrom et al., 1984; Kato et al., 1980). There are several facts indicating that glutamine t R N A suppressor mutations can be targeted at T = C pyrimidine dimers after UV irradiation (Bockrath et al., 1987). Thus, in uvrA6 strains with phr deficiency, there is a specific increase of mutations associated with targeted T
possesses higher amounts of photolyase per cell than a strain with a moderate mutation frequency, and the strain with the highest mutation frequency is defective for phr. These data support the earlier suggestion that D N A photolyase interferes with UV mutagenesis by virtue of binding to the targeting T = C dimer to interrupt mutational D N A synthesis. Recently evidence has been obtained that, in E. coli, D N A photolyase plays varying roles in the dark in the D N A metabolism: stimulation of excision repair in the recA strain, sensitization of excision-deficient strains and reduction of mutations associated with targeting by T = C. Such a dark role of D N A photolyase is not specific for E. coli. Sancer and Smith (1989) have reported that phr + strains of Saccharomyces cerevisiae are more resistant to UV killing than phr-deficient strains in a radl8 background. A photorepair-deficient mutant in Drosophila melanogaster was found to be partially deficient in dark repair and the deficiency was closely linked to a phr mutation (Boyd and Harris, 1987). Thus, in general, in addition to its role in light-dependent repair, D N A photolyase has a role in light-independent repair. As a consequence, if one wants to consider specific repair activities that alter the effect of UV radiation, the use of strains with phr deficiencies can reduce the complexity of interacting effects due to the formation of D N A photolyase and pyrimidine dimer complexes. The alleles we constructed are useful for such a purpose, since the alleles can easily be transferred to the strain of interest just by selecting drug resistance.
~C.
We thank Dr. B.J. Bachmann for providing the strains and Dr. R. Bockrath for critically reading the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Area ' T h e Molecular Mechanism of Photoreception' from the Ministery of Education, Science and Culture of Japan.
It has been demonstrated previously that in strains carrying a large amount of D N A photolyase due to the presence of a multicopy plasmid carrying phr, production of glutamine t R N A suppressor mutations by UV radiation was reduced compared to strains with normal amounts of photolyase (Ruiz-Rubio et al., 1988). Therefore, there is a very good inverse correlation between the frequency of mutations associated with targeting T = C and the amounts of D N A photolyase per cell; the strain with the lowest mutation frequency
Acknowledgements
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