- Email: [email protected]
A mutation in the DNA adenine methylase gene (dam) of Salmonella typhimurium decrease susceptibility to 9-aminoacridine-induced frameshift mutagenesis
131
Mutation Research, 194 (1988) 131-141 DNA Repair Reports Elsevier MTR 06294
A mutation in the D N A adenine methylase gene (dam) of Salmonella typhimurium decreases susceptibility to 9-aminoacridine-induced frameshift mutagenesis Lyndal Ritchie, Denis M. Podger and Ruth M. Hall CSIRO Dioision of Molecular Biology, P.O. Box 184, North Ryde, NSI¥ 2113 (Australia) (Received 30 June 1987) (Revision received 28 March 1988) (Accepted 7 April 1988)
Keywords: DNA adenine methylase gene; dam gene; 9-aminoacridine-induced frameshift mutagenesis
Summary A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the D N A adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopurine (2AP) intermediate between that of the dam + and the D N A adenine methylation-deficit dam-1 strain, and 2AP sensitivity was reversed by introduction of a mutH mutation or of the plasmid pMQ148 (which carries a functional Escherichia coli dam ÷ gene). However, the dam-2 strain is not grossly defective in D N A adenine methylase activity. Whole cell D N A appears full methylated at - G A T C - sites. The levels of 9AA required to induce equivalent levels of frameshift mutagenesis in the dam-2 strain were approximately 2-fold higher than for the dam + strain. Introduction of pMQ148 dam + reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam + strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutations, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam +/pMQ148, d a m - 2 / p M Q 1 4 8 and d a m - 1 / p M Q 1 4 8 strains showed identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced (dam-2) or increased (pMQ148) rate of methylation at the replication fork. The 2AP sensitivity of the dam-2 strain cannot be simply explained. Furthermore, addition of methionine to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutagenesis.
Acridine derivatives such as 9-aminoacridine, proflavine and ICR191 produce predominantly frameshift mutations (Roth, 1974). Studies of the
Correspondence: Dr. R.M. Hall, CSIRO Division of Molecular Biology, P.O. Box 184, North Ryde, NSW 2113 (Australia).
spectrum of forward mutations induced by these compounds in E. coil using the l a d gene on an F ' plasmid or the cI gene of an integrated copy of X phage have determined that the vast majority of mutations are insertion or deletion of a G - C base pair in a monotonous run of 2 or more G - C base pairs (Calos and Miller, 1981; Skopek and
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132 Hutchinson, 1984). This specificity is clearly different from the spectrum of frameshift events induced by proflavine in the bacteriophage T4 (see Ripley and Clark, 1986). The difference probably reflects the fact that T4 encodes many of its own replication functions including the DNA polymerase, and suggests that the specificity of acridine-induced frameshift events is dependent on interactions with the DNA replication system. Several lines of evidence suggest that the site of mutagenic action of acridines is associated with the replication fork. Only exposure of actively growing cells induces high levels of mutations. In synchronized E. coli, treatment of cells with 9AA or ICR191 for short periods led to extensive mutagenesis in a particular gene only when the treatment period corresponded with the time of replication of that gene (Newton et al., 1972). Furthermore, mutants defective in mismatch repair (mutH, mutL routS, and uvrD) or in DNA adenine methylation (dam-) exhibit dramatic increases in susceptibility to the mutagenic effects of 9AA (Skopek and Hutchinson, 1984; Mohn et al., 1984; Ritchie et al., 1986), and DNA adenine methylation-directed mismatch repair is believed to be a postreplicative process. Acridines such s ICR191 which have an alkylating half-mustard sidechain can covalently bind to bases in DNA, and are generally more potent mutagens than their non-alkylating counterparts (Ames and Whitfield, 1966). The covalent lesions are recognized and excised by the uvr + pathway (Newton et al., 1972; Imray and MacPhee, 1976) and also lead to induction of the SOS response (Podger and Hall, 1985; Ohta et al., 1984). As the influence of these pathways can complicate the interpretation of experiments aimed at determining the mechanism of frameshift mutagenesis, the simple acridines such as 9AA appear to provide more suitable models for such studies. This paper reports the characterization of a mutant of S. typhimurium isolated by screening for a reduced response to the mutagenic effects of 9AA. The mutation maps in the DNA adenine methylase (dam) gene, but the DNA of the mutant appears to be fully methylated. Various phenotypic effects o this mutation (dam-2) were compared with those of the wild-type and a DNA adenine methylation-deficient mutant (dam-l).
This comparison does not allow a simple explanation of the effects of the mutant unless it is assumed that the mechanism for sensitivity of dam- strains to the base analogue 2-aminopurine is more complex than has been assumed to date. Materials and methods
Media The defined medium used to detect back-mutations to prototrophy was minimal E salts (Vogel and Bonner, 1956) containing 0.2% (v/v) glucose as carbon source and 1.5% Difco Bacto-agar (Difco Bacto-agar Laboratories, Detroit, MI). Amino acids were supplemented at a concentration of 20 /~g/ml. Bacteria were routinely cultured on complete medium (NYA) containing 0.8% Difco nutrient broth, 0.2% Difco yeast extract, 0.5% NaC1, and 1.5% agar. Overlay agar contained 0.6% Difco Bacto-agar and 0.5% NaC1. Strains The strains of Salmonella typhimurium subline LT2 are listed in Table 1. Introduction of the dam-2 mutation into a non-mutagenized, wild-type repair background (NR5280) was achieved by P22int-3-mediated transduction as described previously for the dam-1 mutation (Ritchie et al., 1986). Either hisC3076, or zec-2 :: TnlO was then introduced by P22 transduction and selection for tetracycline resistance (25 /~g/ml). The plasmid pMQ148 was introduced by transformation (Lederberg and Cohen, 1974). Wild-type, dam-l, and dam-2 strains containing the gale marker linked to zbi-812::Tn10 were constructed by isolating tetracycline-sensitive derivatives of the respective strains NR5068, NR5293, and NR5308, and using these as recipients in transduction crosses with a lysate of P22 grown on strain T N l l l 7 (zbi-812 :: TnlO, galE). Tetracycline-resistant colonies were twice purified, checked for sensitivity to galactose (inability to grow on supplemented minimal E medium containing 1.2% galactose), and those strains carrying a dam mutation were again checked for sensitivity to 2-aminopurine.
133 TABLE 1
S. typhimuriurn LT2 S T R A I N S Strain
Genotype
Source
GW1810 TT3713 NR5280 NR5040 NR5038 NR5265 NR5076 NR5068 NR5293 NR5308
hisG46, rnutHl01:: T n 5 cysG1510 :: T n l 0 , his9707 trpES, cysG1510 : : Tn 10 hisC3076, zec-2 :: Tn l O trpES, uvr-304, hisC3076, zec-2 :: TnlO trpES, uvr-304, hisC3076, zec-2 :: TnlO, dam-1 trpES, uvr-304, hisC3076, zec-2 :: T n 10, dam-2 trpE8, hisC3076, zec-2 :: Tn 10 trpE8, hisC3076, zec-2 : : T n l 0 , dam-1 trpES, hisC3076, zec-2 :: TnlO, darn-2
G.C. Walker J. Roth This study D. MacPhee This study This study This study This study This study This study
TNlll7 NRl167 NRl169 NRll71
zbi-812 :: Tn 10, gale trpE8, hisC3076, zbi-812:: T n 10, gale trp ES, hisC307 6, zbi-812 : : Tn l O, galE, dam-1 trpES, hisC3076, zbi-812 : : Tn l O, galE, dam-2
J. Roth This study This study This study
Mutant isolation The screen procedure used was designed to detect mutants with reduced levels of 9AA-induced reversion of the frameshift marker hisC3076. Cells of NR5038 were mutagenized with Nmethyl-N'-nitro-N'-nitrosoguanidine as described previously (Ritchie et al., 1986). Single colonies were resuspended in 200 /d NYA medium in microtitre trays, and incubated overnight at 37 ° C. Samples (2-5 #1) were transferred using a Titretek Automatic Inoculator (Flow Laboratories) to minimal E plates supplemented with histidine and tryptophan (master plate) and to minimal E plates supplemented with tryptophan and with limiting histidine (0.65/~g/ml) and containing 9AA (Sigma Chemical Co., St. Louis, MO, 300 /~g/plate). A sample of the parent strain was included in each of the 4 comer wells of the tray, and these controis yielded a confluent growth of His ÷ revertants on the 9AA-containing plate. Colonies which showed less than 5 His + revertants were selected and the screen procedure repeated. Colonies which again showed less than 5 His + revertants were screened individually in a well test as follows. 0.1 ml of a stationary phase culture was added to 2 ml overlay agar containing tryptophan and limiting histidine and poured onto minimal E plates. A well was formed in the centre of the plate and 9AA (100 /~g or 25 /~g) added to the well. The plates were incubated for 3 days at 37 ° C, and the
number of His+-revertant colonies was scored. In this assay, the mutant NR5625 again showed significantly lower numbers of His + colonies than the parent strain.
Sensitivity assays Sensitivity to 2-aminopurine (2AP) was determined using well tests. Cells (-- 108) were plated in overlay agar on minimal E supplemented with histidine and tryptophan or on N Y A plates. A well was formed in the centre and 2AP (500 /~g Sigma) was added to the well. Plates were incubated for 2 days at 3 7 ° C and the diameter of the zone of growth inhibition measured. Sensitivity to 9AA determined by plating ~ 200 cells in overlay agar supplemented with histidine and tryptophan and various amount of 9AA. Colonies were counted after 2 days incubation at 37 ° C. To more closely mimic the conditions of the mutation assay 108 filter cells (NR5280) were included with the test cell. The filler cells are unable to grow under these conditions by virtue of a non-revertible autotrophic marker (cysG::
TnlO). Assay for DNA methylation Whole cell or plasrnid D N A was isolated by a rapid isolation procedure (Davis et al., 1980) and digested with Sau3AI, MboI and DpnI (New England Biolabs, Inc., Beverly, MA). D N A was
134
electrophoresed in 0.8% agarose gel, stained with ethidium bromide and photographed.
TABLE 2
Mutagenicity assays Mutation assays were carried out according to the published plate-test procedures (Ames et al., 1975). A small volume of an aqueous stock solution of 9AA or ICR191 (Polysciences Inc., Warrington, PA) (no greater than 100 /~l/p!ate ) and 100 /xl of fresh stationary-phase bacterial culture grown in NYA medium (ca. 2 x 108 cells) were added to 2.0 ml of overlay agar held at 46 o C. The contents of the tubes were mixed and poured over the surface of agar plates containing 25 ml of minimal E selection medium supplemented with 20 # g / m l L-tryptophan and 0.8 # g / m l L-histidine. Overlay agar was allowed to solidify and the plates were incubated at 37 ° C for 3 days. At the end of the incubation period back-mutation yields were scored at least in duplicate with the aid of an Artek 880 automatic counter (Artek Systems Corp., Farmingdale, NY). All experiments were repeated 3 times. The doses of 9AA and ICR 191 used in these assays were shown to have no effect on cell survival.
Other conditions
Assays for TnlO activity For measurement of Tnl0-mediated rearrangements extending into the gal region, cells (approximately 2 x 105) incorporated in 2 ml of overlay agar were poured onto supplemented minimal E medium and incubated overnight for aproximately 15 h. Plates were overlayed with a mixture of 2 ml of 2 x overlay agar and 1.5 ml of 20% D-galactose, and i n c u b a t e d for 2 days. Galactose-resistant colonies were counted. To test for retention of TnlO, colonies were purified by streaking twice for single colonies on supplemented minimal E medium containing 1.2% Dgalactose, and tested for tetracycline resistance by streaking on N Y A plates containing 25 /xg/ml tetracycline. For all strains 65-75% of colonies were tetracycline sensitive. Results
(1) Isolation and characterization of NR5076. The mutant NR5076 was isolated after treatment of N R 5038 (uvr-304, trpES, hisC3076) with
SENSITIVITY TO 2-AMINOPURINE
m u t H l O 1 :: T n 5 b pMQ148 dam + b Methionine c
Zone diameter a (mm) dam +
dam- 1
d a m -2
10 9 10 12
38 9 9 37
21 9 10 12.5
a Diameters are averages of 3 determinations. b m u t H l O 1 : : T n 5 o r p M Q 1 4 8 d a m + w a s i n t r o d u c e d i n t o the s t r a i n (see M e t h o d s ) . c M e t h i o n i n e (45 # g / m l ) w a s a d d e d to the plates.
N-methyl-N'-nitro-N'nitrosoguanidine, and detected in a screen for mutants with reduced levels of reversion of the hisC3076 frameshift marker by 9-aminoacridine. Mutants isolated in this way were screened for sensitivity to the lethal effects of several agents including 2-aminopurine. As 2AP sensitivity is characteristic of dam- mutants in E. coli (Glickman et al., 1978), and the 2AP sensitivity of dam- mutants is reversed by the presence of mutations in the genes of the mismatch repair pathway (McGraw and Marinus, 1980; Glickman and Radman, 1980), the mutHlOl::Tn5 allele was introduced into all mutants showing 2AP sensitivity. For 2 mutants (NR5265 and NR5076) 2AP sensitivity was reversed by the mutH allele (Table 2). NR5265 dam-1 has been shown to be completely devoid of D N A adenine methylation, and the mutation (dam-l) maps in the D N A adenine methylase gene (Ritchie et al., 1986). NR5076 showed a level of 2AP sensitivity intermediate between that of NR5038 (dam +) and of NR5265 (dam-l) (see Table 2). Introduction of the plasmid pMQ148 which contains a functional E. coli dam ÷ gene also reversed the 2AP sensitivity of NR5076. As pMQ148 carries only the functional E. coli D N A adenine methylase gene and reverses the phenotypic characteristics of dammutants in E. coli (Arraj and Marinus, 1983) and S. typhimurium (Ritchie et al., 1986), this result suggests that the mutation in NR5076 is located in the dam gene. The 2AP sensitivity was cotransducible with cysG (44.5% cotransduction) which confirms the location of the mutation (dam-2) in
135 the D N A adenine methylase gene (Marinus, 1973; Ritchie et al., 1986). An interesting feature of the mutant NR5076 (dam-2) is the reversal of 2AP sensitivity by addition of methionine to the medium (see Table 2). This effect is not observed in strains carrying the dam-1 mutation, nor is the level of 2AP sensitivity of dam ÷ strains reduced by methionine. The dam-2 mutation was introduced into a non-mutagenized background, and this strain NR5308 was used in all further experiments.
(2) DNA adenine methylation in NR5308 The methylation status of adenine residues in the sequence - G A T C - was investigated using the restriction enzymes Sau3A, MboI and DpnI, all of which recognize this sequence. Sau3A cleaves both methylated and unmethylated sites, MboI cleaves only unmethylated sites and DpnI cleaves only methylated sites. Cellular D N A was isolated from NR5308 (dam-2) and digested with the restriction enzymes (Sau3A, DpnI and MboI). The D N A was completely digested by Sau3A and by DpnI and not detectably digested by MboI (not shown). These results suggest that the majority of - G A T C - sites are methylated in NR5308 and thus that the dam-2 mutation does not lead to substantial inactivation of the D N A adenine methylase. To obtain a more sensitive assay for the presence of unmethylated sites in D N A from NR5308, the plasmid pBR322 was introduced and plasmid D N A isolated from an unamplified culture. This D N A was completely digested by Sau3A and DpnI (not shown). Some nicking and cleavage by MboI was observed in plasmid D N A isolated from both NR5068 (dam ÷) and NR5308 (dam-), suggesting the presence of a single unmethylated or hemimethylated site in a small number of molecules, but no difference between the strains was detectable. Thus the dam-2 mutation does not lead to a detectable reduction in the level of methylation of - G A T C - sequences in the DNA. (3) Spontaneous mutability An elevated spontaneous mutation frequency is characteristic of dam- mutants (Bale et al., 1979; Glickman, 1979). Spontaneous reversion of the
100
100
~o
survival
200
9AA (ugl01ate) 30O 40O 500
600
,o
[
Dam + d,.am-2
1
Fig, 1.9-Aminoacridinesensitivityof dam÷, dare-1 and dam-2 strains. 9-Aminoacridinewas included with 500 cells and filler cells in overlay agar. A, NR5068 dam÷;@,NR5293 darn-l; I, NR5308 dam-2. Values are averages of 3 independent duplicate determinations.
base substitution marker trpE8 and the frameshift marker hisC3076 in the dam-2 strain was not significantly different (21.3 Trp ÷ and 5.9 His + colonies/plate) from the dam ÷ strain (21.0 Trp ÷ and 5.8 His + colonies/plate). Spontaneous reversion of these markers is elevated 5- and 20-fold respectively in the dam-1 strain (Ritchie et al., 1986).
(4) 9-Aminoacridine sensitioity The procedure used to isolate the dam-1 and dam-2 strain involved screening for reduced numbers of 9AA-induced His + revertants of the hisC3076 mutation. In the case of the dam-1 strain we have reported that 9AA mutagenesis is in fact dramatically increased (Ritchie et al., 1986). The detection of this strain may have resulted from the increased sensitivity of the dam-1 strain to the lethal effects of 9AA (Fig. 1). Sensitivity of dam-1 strains to 9AA is reversed by introduction of the mutHl05 :: T n 5 mutation or the dam + plasmid pMQ148 (not shown). However, the dam-2 strain is not significantly sensitive to 9AA (Fig. 1).
136
IT
1500
|
/ / HIs' 1000
/
l /
o.m+
dam-1
7
dam
C')dam-I/ / dam-,2/ DM--"Q"~4 ~ / DMQ148/ -Dam+/
/
/
colonies per plate 500
50
loo
9AA (pg/plate)
150
200
250
Fig. 2. 9-Aminoacridine-induced reversion of hisC3076 in dam +, dam.1 and dam-2 strains with and without the plasmid pMQ148 dam +. 9-Aminoacridine was included with 2 - 5 × 1 0 ~ cells in 2 ml of overlay agar on minimal plates with limiting histidine. A, NR5068, O, NR5293 dam-l; I , NR5308 dam-2; A, NR5068 dam+/pMQ148; o, NR5293 dam-1/pMQ148; D, NR5308 dam-2~ pMQ148. Values are averages of 3 independent duplicate determinations.
(5) Frameshift mutagenesis
The dose-response curves for 9AA-induced reversion of the hisC3076 marker in the dam-2 and dam ÷ strains, using plates assays, are shown in Fig. 2. The dose response for the dam-1 strain is also shown for comparison. The 2-component dose-response curve observed in dam + strains presumably results from efficient repair 9AA-induced errors up to a threshold dose beyond which the capacity of the mismatch repair system to correct errors is exceeded and mutations are induced. In the dam-2 strain the threshold dose is increased approximately 2-fold indicating a greater capacity for error correction in this strain. Addition of methionine to the medium does not reverse this effect (not shown). Reduced susceptibility of the dam-2 strain to the mutagenic effects of 9AA was also observed when the dose response to 9AA is assayed following treatment of a log-phase culture with various doses of 9AA for 30 min. The number of His ÷ revertants induced per #g 9AA was 10.2 for NR5038 (dam +) and 2.6 for NR5076 (dam-2). The dose-response curves for dam +, dam-1 and dam-2 strains carrying the dam + plasmid pMQ148 are also shown in Fig. 2. All 3 plasmidcontaining strains exhibit similar dose-response
curves, and the threshold dose for mutation induction is about 2-fold lower than for the dam ÷ strain without the plasrnid. This result implies a reduced capacity for mismatch repair in the presence of the multicopy dam ÷ plasmid.
2000I | 15001
/
,~ Dam*/pMO148 laam-~21pM0148 //
'1' ° ' " '
I I fo..._,/,Mo,,, III
I / I /
""
::°":oooI # //
'"'"
5
0
o lO
f/ 0
///
..-°Lm:
~
40 60 80 100 ICR191(pg/plale)
150
Fig. 3. ICR191-induced reversion of hisC3076 in dam +, dam-I and dam-2 strains with and without the plasmid pMQ148 dam +. Strains used are described in the legend to Fig. 2. Values are averages of 3 independent duplicate determinations.
137
The dose-response curves for ICR191-induced reversion of hisC3076 in darn +, dam-1 and dam-2 strains with and without pMQ148 are shown in Fig. 3. The dose response with ICR191 in the darn + strain differs from that observed with 9AA, and is essentially linear, though a small threshold dose effect is observed. However, mismatch repair also plays a significant role in avoidance of ICR191-induced errors as evidenced by the dramatic increase in slope observed for the darn-1 strain. In contrast to the results obtained for 9AA, the darn-2 mutation has little or no effect on induction of frameshift errors by ICR191. ICR191 binds covalently to DNA, forming lesions which can be excised by the enzymes of the uorABC pathway (Newton et al., 1972; Imray and MacPhee, 1976). However in the uvr-304 background, the darn + and darn-2 strains also show identical dose responses (not shown), though substantially lower doses are required for equivalent levels of mutagenesis. Thus removal of mutagenic lesions by the uvrABC pathway is not responsible for the failure to detect a difference between the dam + and darn-2 strains when ICR191 is used as the mutagen. Introduction of the multicopy plasmid pMQ148 darn + reverses the effect of the darn-1 mutation and leads to a 2-fold increase in the slope for both darn ÷ and darn-2 strains (Fig. 3). Thus it appears that the dam-2 mutation has no effect on the repair of frameshift errors induced by ICR191, but that overproduction of the E. coli DNA adenine methylase by pMQ148 reduces the amount of error correction. (6) Transposon activity
Mutants deficient in DNA adenine methylase also exhibit increased T n l O and Tn5 transposon activity (Arraj and Marinus, 1983; Lundblad and Kleckner, 1984; Roberts et al., 1985). For T n l O this effect has been shown to result from the influence of the methylation status of particular - G A T C - sites on the expression of transposition functions (Roberts et al., 1985). Tnl0-promoted deletions and rearrangements were assayed by induction of galactose resistance in strains carrying galE and a closely situated zbi-812 :: T n l O insertion (Table 3). Tnl0-promoted deletions and rearrangements which extend into the gal operon
TA B LE 3
TnlO A C T I V I T Y IN dam +, darn-1 A N D dam-2 S T R A I N S Allele
Tn I 0-promoted rearrangements gale zbi-812 :: TnlO G a l r c o l o n i e s / p l a t e a
+
77 715 82
dam-1 darn-2
a Values are averages of 3 independent determinations. The numbers of cells per plate, before selection for G a l r was applied, was identical for the 3 strains.
reverse the sensitivity to galactose conferred by the gale mutation. No difference was observed between the darn + and darn-2 strains, while a 10-fold increase was observed in the darn-1 strain. The increased T n l O activity in the darn-1 strain is consistent with observations in E. coli d a m strains. Discussion The dam-2 mutant described here differs in several respects from the DNA adenine methylation-deficient (darn-) mutants which have been previously described. Most strikingly the DNA of the dam-2 mutant appears to be fully methylated indicating that the methylase is not grossly defective in methylation activity. The many phenotypic effects of darn- mutations have been reviewed by Marinus (1984). For several of these phenotypes (increased spontaneous mutability, increased Tn 10 activity, increased sensitivity to 9AA), the darn-2 strain resembles the wild-type dam + strain. In contrast, dam-2 resembles darn-1 in conferring sensitivity to 2AP and in that this sensitivity is reversed by a mutation in one of the genes (mutH) of the mismatch repair pathway. However the 2AP sensitivity of the dam-2 strain is lower than that of the dam-1 strain and is reversed by addition of methionine to the assay medium. Finally the susceptibility to mutagenesis by 9AA is reduced 2-fold by dam-2 but increased substantially by darn-1. The proposal that DNA adenine methylation provides the mismatch repair system with a mechanism for strand discrimination is now well supported by experiments using in vitro constructed heteroduplexes of bacteriophage h and M13 DNA
138 which contain a single mismatch (Dohet et al., 1985; Kramer et al., 1984; Pukkila et al., 1983; Radman et al., 1985) or single base additions or deletions (Dohet et al., 1986; Radman et al., 1985). When only 1 strand of the heteroduplex is methylated, the unmethylated strand is preferentially repaired. When neither strand is methylated, repair occurs without strand discrimination, and when both strands are methylated no repair is observed. For the E. coli chromosome, mismatch repair is believed to be a post-replicative process which rectifies errors generated during replication and not corrected by proofreading functions. It has been proposed that the newly synthesized strand is transiently unmethylated at - G A T C - sites in the D N A at the replication fork and that these hemimethylated sites provide the strand discrimination mechanism. This notion is supported by evidence that the newly synthesized strand is undermethylated (Marinus, 1976; Lyons and Schendel, 1984). Furthermore, the spontaneous mutation frequency is substantially increased in d a m - mutants of E. coli (Bale et al., 1979; Glickman, 1979) and in S. typhimurium (Ritchie et al., 1986) as well as in mismatch repair-deficient mutL, H, S and uorD mutants (Rydberg, 1978; Shanabruch et al., 1981; Glickman and Radman, 1980) and the spectrum of spontaneous mutations is similar in d a m - and rout- strains (Glickman, 1979; Leong et al., 1986). Comparison of the dose-response curves for 9AA-induced frameshift reversion events in d a m and d a m - strains of E. coli (Mohn et al., 1984) and S. typhimurium (Ritchie et al., 1986) indicate that in dam ÷ strains the majority of 9AA-induced frameshifts errors are accurately and efficiently repaired by the adenine methylation-instructed mismatch repair pathway. As mutation induction occurs at low 9AA doses in the d a m - strain, the virtual absence of induced revertants in the dam ÷ strain at 9AA doses below 100 ~tg 9 A A / p l a t e can be attributed to complete repair of induced lesions. At higher doses the capacity of the repair pathways becomes saturated and mutation induction is observed. As methylation-instructed repair is primarily a post-replicative process, this observation also implies that the majority of 9AA-induced errors occur during the replication process. The increased levels of 9AA required to induce
frameshift errors in the dam-2 strain described here can most simply be explained by postulating that in dam-2 the methylation of hemimethylated - G A T C - sites at the replication fork occurs more slowly than in the dam ÷ strain, allowing a longer period in which the mismatch repair system can discriminate between strands and preferentially repair the newly synthesized strand. A similar explanation can account for the reduced levels of 9AA required for mutagenesis in dam ÷ and darn-2 strains carrying the multicopy pMQ148 dam ÷ plasmid. Overproduction of the D N A adenine methylase should increase the rate of methylation at the form and thus reduce the amount of mismatch repair which can occur. Indeed it has been shown that the spontaneous mutation frequency of dam ÷ strains is elevated by introduction of dam ÷ plasmids which overproduce the D N A adenine methylase 20-50-fold (Herman and Modrich, 1981) or 300-500-fold (Marinus et al., 1984). However a plasmid which results in 15-fold over-expression is without effect (Marinus et al., 1984). It is possible that for spontaneous mutations the number of replication errors is sufficiently low that the rate of methylation does not become rate limiting until very high levels of over-expression are achieved. Moreover the basal level of methylase activity may vary considerably in different strains. We have found that the spontaneous reversion of the frameshift marker hisC3076 and the base substitution marker trpE8 is unaffected in the dam-2 strain and also unaffected by the presence of pMQ148 plasmid which includes the dam gene in a pBR322 vector. However, using 9AA-induced mutagenesis as an assay could be expected to be far more sensitive to small variations in the rate of methylation as many more mismatch-correctable lesions are present in this case. When ICR191 was used as the mutagen several differences from 9AA are observed. The dose response does not exhibit the clear threshold, seen with 9AA. Nonetheless, the dramatic increase in slope observed with the dam-1 strain indicates that mismatch repair is responsible for correction of the majority of ICR191-induced errors. Though the multicopy dam ÷ plasmid increases the slope of the dose-response curve, no difference between the dam + and dam-2 strains is observed. We are
139 unable to offer a simple explanation for these findings. The proposal that methylation at the fork is slower in the dam-2 strain fails to explain the 2AP-sensitive phenotype of the dam-2 strain. To date the 2AP sensitivity of d a m - strains has been explained by proposing that because the D N A is unmethylated, strand discrimination cannot occur and breaks are introduced into both strands by the enzymes of the mut pathway in response to the presence of 2AP in the DNA. Sensitivity then reflects the introduction of double-strand breaks. The presence of nicks in the D N A is supported by the fact that 2AP induces the SOS response, d a m but not in dam + strains (Bebenek and Janion, 1985; Craig et al., 1984), and the involvement of the mismatch repair pathway is supported by the finding that mutH, mutL and mutS mutations reverse both the 2AP sensitivity (Glickman and Radman, 1980; McGraw and Marinus, 1980) and the 2AP SOS-inducing ability (Craig et al., 1984; Bebenek and Janion, 1985) of d a m - strains. More direct evidence for mismatch repair-induced double-strand breaks in unmethylated D N A has recently been reported (Doutriaux et al., 1986; Wang and Smith, 1986). However, an indication that this explanation for sensitivity of d a m - strains to base analogues is somewhat simplistic comes from studies on the adenine analogues 2-amino-N6-hydroxyadenine and 2-amino-N6-methoxyadenine. Mutagenesis by these analogues is elevated in d a m - strains, but d a m - strains are sensitive only to the lethal effects of 2-amino-N6-hydroxy adenine (Bebenek and Janion, 1983). Furthermore, both analogues resemble 2AP in that they induce the SOS response in d a m - cells, but not in d a m - r o u t - cells (Bebenek and Janion, 1985). In the case of the dam-2 mutant described here, the D N A is essentially fully methylated. Introduction of nicks in both strands leading to doublestrand breaks cannot be invoked to explain the 2AP sensitivity, as the parental strand is methylated and nicks should only be introduced into the newly synthesized unmethylated strand. However the mismatch repair pathway appears to be involved, as a dam-2 m u t H : : T n 5 strain is no longer 2AP sensitive. Furthermore, the D N A adenine methylase is also involved as the presence of the E. coli dam + methylase gene on a multicopy
plasmid also reverse 2AP sensitivity. A possible explanation is that the dam-2 methylase is particularly sensitive to the cellular level of its substrate methyl donor, S-adenosylmethionine (SAM), and that 2AP affects the adenine nucleotide pools, thus reducing the SAM concentration and leading to extensive demethylation of D N A , 2AP-induced cell death would then occur by a mechanism akin to that which occurs in d a m - cells. If addition of methionine restores the SAM levels to normal, this would account for the reversal of the 2AP sensitivity of the dam-2, but not the dam-1 strain, by addition of methionine to the medium. The S. typhirnurium dam-1 strain exhibits sensitivity of 9AA, and this effect is also reversed by introduction of a m u t H mutation or the dam + plasmid. However the mechanism of 9AA sensitivity cannot be identical to that for 2AP sensitivity, as the dam-2 strain is not significantly more sensitive than the wild type. The introduction of nicks on either strand at the site of 9AA-induced errors is in this case a plausible explanation for 9AA sensitivity in the dam-1 strain. However, 9AA does not appear to induce the SOS response in d a m - E. coli cells (Quillardet and Hofnung, personal communication). Further analysis of the dam-2 mutant is clearly required to clarify some of the issues raised in this study. In particular direct measurement of the degree of methylation at the fork, or a study of the properties of the purified methylase is required to determine if the rate of D N A adenine methylation at the replication fork is reduced in this mutant.
Acknowledgements We thank Trish Hayes for competent technical assistance.
References Ames, B.N., and H.J. Whitfield Jr. (1966) Frameshift mutagenesis in Salmonella, Cold Spring Harbor Symp. Quant. Biol., 31, 221-225. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsometest, Mutation (Res., 31, 347-364. Arraj, J.A., and M.G. Marinus (1983) Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam + gene, J. Bacteriol., 153, 562-565.
140 Bale, A., M. d'Alarcao and M.G. Marinus (1979) Characterization of DNA adenine methylation mutants of Escherichia coil K12, Mutation Res., 59, 157-165. Bebenek, K., and C. Janion (1983) Involvement of the mismatch repair system in base analogne-induced mutagenesis, Mol. Gen. Genet., 1991, 276-281. Bebenek, K., and C. Janion (1985) Ability of base analogues to induce the SOS response: effect of a dam mutation and mismatch repair system, Mol. Gen. Genet., 201, 519-524. Calos, M.P., and J.H. Miller (1981) Genetic and sequence analysis of frameshift mutations induced by ICR191, J. Mol. Biol., 153, 39-66. Craig, R.J., J.A. Arraj and M.G. Marinus (1984) Induction of damage inducible (SOS) repair in dam mutants of Escherichia coil exposed to 2-aminopurine, Mol. Gen. Genet., 194, 539-540. Davis, R.W., D. Botstein and J.R. Roth (1980) A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Dohet, C., R. Wagner and M. Radman (1985) Repair of defined single base-pair mismatches in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 82, 503-505. Dohet, C., R. Wagner and M. Radman (1986) Methyl-directed repair of frame,shift mutations in heteroduplex DNA, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3395-3397. Doutriaux, M-P., R. Wagner and M. Radman (1986) Mismatch stimulated killing, Proc. Natl. Acad. Sci. (U.S.A.), 83, 2576-2578. Glickrnan, B.W. (1979) Spontaneous mutagenesis in Escherichia coil strains lacking 6-methyladenine residues in their D N A , Mutation Res., 61, 153-162. Glickman, B.W., and M. Radman (1980) Escherichia coil mutator mutants deficient in rnethylation-instructed D N A mismatch correction, Proc. Natl. Acad. SCI. (U.S.A.), 77, 1063-1067. Glickman, B.W., P. van den Elsen and M. Radman (1978) Induced mutagenesis in dam- mutants of Escherichia coil, A role of 6-methyladenine residues in mutation avoidance, Mol. Gen. Genet., 163, 307-312. Herman, G.E., and P. Modrich (1981) Escherichia coli K-12 clones that overproduce dam methylase are hypermutable, J. Bacteriol., 145, 64,*-646. Imray, P., and D.G. MacPhec (1976) Spontaneous and induced mutability of frameshift strains of Salmonella typhimurium carrying uvrB and poL4 mutations, Mutation Res., 34, 35-42. Kramer, B., W. Kramer and H.-J. Fritz (1984) Different b a s e / base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coil, Cell, 38, 879-887. Lederberg, E.M., and S.N. Cohen (1974) Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid, J. Bacteriol., 119, 1072-1074. Leong, P.-M., H.C. Hsia and J.H. Miller (1986) Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coil, J. Bacteriol., 168, 412-416. Lundblad, V., and N. Kleckner (1984) Mismatch repair muta-
tions of Escherichia coli K12 enhance transposon excision, Genetics, 109, 3-19. Lyons, S.M., and P.F. Schendel (1984) Kinetics of methylation in Escheriehia coli K-12, J. Bacteriol., 159, 421-423. McGraw, B.R., and M.G. Marinus (1980) Isolation and characterization of Dam + revertants and suppression mutants that modify secondary phenotypes of dam-3 strains of Escherichia coli K12, Mol. Gen. Genet., 178, 309-315. Marinus, M.G. (1973) Location of methylation genes on the Escheriehia coli K-12 genetic map, Mol. Gen. Genet., 127, 47-55. Marinus, M.G. (1976) Adenine methylation of Okazaki fragments in Eseherichm coli, J. Bacteriol., 128, 853-854. Marinus, M.G. (1984) Methylation of procaryotic DNA, in: A. Razin, H. Cedar and A. Riggs (Eds.), DNA Methylation, Biochemistry and Biological Significance, Springer, New York, pp. 81-109. Marinus, M.G., A. Poteete and J.A. Arraj (1984) Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K12, Gene, 28, 123-125. Mohn, G.R., P.R.M. Kerklaan, A.A. van Zeeland, J. Ellenberger, R.A. Baan, P.H.M. Lohman and F.W. Pons (1984) Methodologies for the determination of various genetic effects in permeable strains of E. coli K-12 differing in DNA repair capacity, Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal mediated assays, Mutation Res., 125, 153-184. Newton, A., D. Masys, E. Leonardi and D. Wygal (1972) Association of induced frameshift mutagenesis and DNA replication in Escherichia coli, Nature (London), New Biol., 236, 19-22. Ohta, T., N. Nakamura, M. Moriya, Y. Shirasu and T. Kada (1984) The SOS function-inducing activity of chemical mutagens in Escherichia coil, Mutation Res., 131,101-109. Podger, D.M., and R.M. Hall (1985) Induction of the SOS response by ICR191 does not influence frameshift mutagenesis at the hisC3076 marker of Salmonella typhimurium, Mutation Res., 142, 87-91. Pukkila, P.J., J. Peterson, G. Herman, P. Modrich and M. Meselson (1983) Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coil, Genetics, 104, 571-582. Radman, M., C. Dohet, M. Jones, M.P. Doutriaux, F. Laengle-Roualt, G. Maenhaut-Michel and R. Wagner (1985) Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli, Biochemie, 67, 745-752. Ripley, L.S., and A. Clark (1986) Frameshift mutations produced by proflavine in bacteriophage T4: specificity within a hotspot, Proc. Natl. Acad. Sci. (U.S.A.), 83, 6954-6958. Ritchie, L.J., R.M. Hall and D.M. Podger (1986) A mutant Salmonella typhimurium LT2 deficient in DNA adenine methylation, J. Bacteriol., 167, 420-422. Roberts, D., B.C. Hoopes, W.R. McLure and N. Kleckner (1985) IS10 transposition is regulated by DNA adenine methylation, Cell, 43, 117-130. Roth, J.R. (1974) Frameshift mutations, Annu. Rev. Genet., 8, 319-346.
141
Rydberg, B. (1978) Bromouracil mutagenesis and mismatch repair in mutation strains of Escherichia coli, Mutation Res., 52, 11-24. Shanabruch, W.G., I. Behlau and C.G. Walker (1981) Spontaneous Salmonella typhimurium LT2 generated by insertion of transposable elements, J. Bacteriol., 147, 827-835. Skopek, T.R., and F. Hutchinson (1984) Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191, Mol. Gen. Genet., 195, 418-423.
Vogel, H.J., and D.M. Bonner (1956) Acetyiornithase of Escherichia coli: partial purification and some properties, J. Biol. Chem., 218, 97-106. Wang, T.-C.V., and K.C. Smith (1986) Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair, J. Bacteriol., 165, 1023-1025.
Mutation Research, 194 (1988) 131-141 DNA Repair Reports Elsevier MTR 06294
A mutation in the D N A adenine methylase gene (dam) of Salmonella typhimurium decreases susceptibility to 9-aminoacridine-induced frameshift mutagenesis Lyndal Ritchie, Denis M. Podger and Ruth M. Hall CSIRO Dioision of Molecular Biology, P.O. Box 184, North Ryde, NSI¥ 2113 (Australia) (Received 30 June 1987) (Revision received 28 March 1988) (Accepted 7 April 1988)
Keywords: DNA adenine methylase gene; dam gene; 9-aminoacridine-induced frameshift mutagenesis
Summary A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the D N A adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopurine (2AP) intermediate between that of the dam + and the D N A adenine methylation-deficit dam-1 strain, and 2AP sensitivity was reversed by introduction of a mutH mutation or of the plasmid pMQ148 (which carries a functional Escherichia coli dam ÷ gene). However, the dam-2 strain is not grossly defective in D N A adenine methylase activity. Whole cell D N A appears full methylated at - G A T C - sites. The levels of 9AA required to induce equivalent levels of frameshift mutagenesis in the dam-2 strain were approximately 2-fold higher than for the dam + strain. Introduction of pMQ148 dam + reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam + strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutations, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam +/pMQ148, d a m - 2 / p M Q 1 4 8 and d a m - 1 / p M Q 1 4 8 strains showed identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced (dam-2) or increased (pMQ148) rate of methylation at the replication fork. The 2AP sensitivity of the dam-2 strain cannot be simply explained. Furthermore, addition of methionine to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutagenesis.
Acridine derivatives such as 9-aminoacridine, proflavine and ICR191 produce predominantly frameshift mutations (Roth, 1974). Studies of the
Correspondence: Dr. R.M. Hall, CSIRO Division of Molecular Biology, P.O. Box 184, North Ryde, NSW 2113 (Australia).
spectrum of forward mutations induced by these compounds in E. coil using the l a d gene on an F ' plasmid or the cI gene of an integrated copy of X phage have determined that the vast majority of mutations are insertion or deletion of a G - C base pair in a monotonous run of 2 or more G - C base pairs (Calos and Miller, 1981; Skopek and
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
132 Hutchinson, 1984). This specificity is clearly different from the spectrum of frameshift events induced by proflavine in the bacteriophage T4 (see Ripley and Clark, 1986). The difference probably reflects the fact that T4 encodes many of its own replication functions including the DNA polymerase, and suggests that the specificity of acridine-induced frameshift events is dependent on interactions with the DNA replication system. Several lines of evidence suggest that the site of mutagenic action of acridines is associated with the replication fork. Only exposure of actively growing cells induces high levels of mutations. In synchronized E. coli, treatment of cells with 9AA or ICR191 for short periods led to extensive mutagenesis in a particular gene only when the treatment period corresponded with the time of replication of that gene (Newton et al., 1972). Furthermore, mutants defective in mismatch repair (mutH, mutL routS, and uvrD) or in DNA adenine methylation (dam-) exhibit dramatic increases in susceptibility to the mutagenic effects of 9AA (Skopek and Hutchinson, 1984; Mohn et al., 1984; Ritchie et al., 1986), and DNA adenine methylation-directed mismatch repair is believed to be a postreplicative process. Acridines such s ICR191 which have an alkylating half-mustard sidechain can covalently bind to bases in DNA, and are generally more potent mutagens than their non-alkylating counterparts (Ames and Whitfield, 1966). The covalent lesions are recognized and excised by the uvr + pathway (Newton et al., 1972; Imray and MacPhee, 1976) and also lead to induction of the SOS response (Podger and Hall, 1985; Ohta et al., 1984). As the influence of these pathways can complicate the interpretation of experiments aimed at determining the mechanism of frameshift mutagenesis, the simple acridines such as 9AA appear to provide more suitable models for such studies. This paper reports the characterization of a mutant of S. typhimurium isolated by screening for a reduced response to the mutagenic effects of 9AA. The mutation maps in the DNA adenine methylase (dam) gene, but the DNA of the mutant appears to be fully methylated. Various phenotypic effects o this mutation (dam-2) were compared with those of the wild-type and a DNA adenine methylation-deficient mutant (dam-l).
This comparison does not allow a simple explanation of the effects of the mutant unless it is assumed that the mechanism for sensitivity of dam- strains to the base analogue 2-aminopurine is more complex than has been assumed to date. Materials and methods
Media The defined medium used to detect back-mutations to prototrophy was minimal E salts (Vogel and Bonner, 1956) containing 0.2% (v/v) glucose as carbon source and 1.5% Difco Bacto-agar (Difco Bacto-agar Laboratories, Detroit, MI). Amino acids were supplemented at a concentration of 20 /~g/ml. Bacteria were routinely cultured on complete medium (NYA) containing 0.8% Difco nutrient broth, 0.2% Difco yeast extract, 0.5% NaC1, and 1.5% agar. Overlay agar contained 0.6% Difco Bacto-agar and 0.5% NaC1. Strains The strains of Salmonella typhimurium subline LT2 are listed in Table 1. Introduction of the dam-2 mutation into a non-mutagenized, wild-type repair background (NR5280) was achieved by P22int-3-mediated transduction as described previously for the dam-1 mutation (Ritchie et al., 1986). Either hisC3076, or zec-2 :: TnlO was then introduced by P22 transduction and selection for tetracycline resistance (25 /~g/ml). The plasmid pMQ148 was introduced by transformation (Lederberg and Cohen, 1974). Wild-type, dam-l, and dam-2 strains containing the gale marker linked to zbi-812::Tn10 were constructed by isolating tetracycline-sensitive derivatives of the respective strains NR5068, NR5293, and NR5308, and using these as recipients in transduction crosses with a lysate of P22 grown on strain T N l l l 7 (zbi-812 :: TnlO, galE). Tetracycline-resistant colonies were twice purified, checked for sensitivity to galactose (inability to grow on supplemented minimal E medium containing 1.2% galactose), and those strains carrying a dam mutation were again checked for sensitivity to 2-aminopurine.
133 TABLE 1
S. typhimuriurn LT2 S T R A I N S Strain
Genotype
Source
GW1810 TT3713 NR5280 NR5040 NR5038 NR5265 NR5076 NR5068 NR5293 NR5308
hisG46, rnutHl01:: T n 5 cysG1510 :: T n l 0 , his9707 trpES, cysG1510 : : Tn 10 hisC3076, zec-2 :: Tn l O trpES, uvr-304, hisC3076, zec-2 :: TnlO trpES, uvr-304, hisC3076, zec-2 :: TnlO, dam-1 trpES, uvr-304, hisC3076, zec-2 :: T n 10, dam-2 trpE8, hisC3076, zec-2 :: Tn 10 trpE8, hisC3076, zec-2 : : T n l 0 , dam-1 trpES, hisC3076, zec-2 :: TnlO, darn-2
G.C. Walker J. Roth This study D. MacPhee This study This study This study This study This study This study
TNlll7 NRl167 NRl169 NRll71
zbi-812 :: Tn 10, gale trpE8, hisC3076, zbi-812:: T n 10, gale trp ES, hisC307 6, zbi-812 : : Tn l O, galE, dam-1 trpES, hisC3076, zbi-812 : : Tn l O, galE, dam-2
J. Roth This study This study This study
Mutant isolation The screen procedure used was designed to detect mutants with reduced levels of 9AA-induced reversion of the frameshift marker hisC3076. Cells of NR5038 were mutagenized with Nmethyl-N'-nitro-N'-nitrosoguanidine as described previously (Ritchie et al., 1986). Single colonies were resuspended in 200 /d NYA medium in microtitre trays, and incubated overnight at 37 ° C. Samples (2-5 #1) were transferred using a Titretek Automatic Inoculator (Flow Laboratories) to minimal E plates supplemented with histidine and tryptophan (master plate) and to minimal E plates supplemented with tryptophan and with limiting histidine (0.65/~g/ml) and containing 9AA (Sigma Chemical Co., St. Louis, MO, 300 /~g/plate). A sample of the parent strain was included in each of the 4 comer wells of the tray, and these controis yielded a confluent growth of His ÷ revertants on the 9AA-containing plate. Colonies which showed less than 5 His + revertants were selected and the screen procedure repeated. Colonies which again showed less than 5 His + revertants were screened individually in a well test as follows. 0.1 ml of a stationary phase culture was added to 2 ml overlay agar containing tryptophan and limiting histidine and poured onto minimal E plates. A well was formed in the centre of the plate and 9AA (100 /~g or 25 /~g) added to the well. The plates were incubated for 3 days at 37 ° C, and the
number of His+-revertant colonies was scored. In this assay, the mutant NR5625 again showed significantly lower numbers of His + colonies than the parent strain.
Sensitivity assays Sensitivity to 2-aminopurine (2AP) was determined using well tests. Cells (-- 108) were plated in overlay agar on minimal E supplemented with histidine and tryptophan or on N Y A plates. A well was formed in the centre and 2AP (500 /~g Sigma) was added to the well. Plates were incubated for 2 days at 3 7 ° C and the diameter of the zone of growth inhibition measured. Sensitivity to 9AA determined by plating ~ 200 cells in overlay agar supplemented with histidine and tryptophan and various amount of 9AA. Colonies were counted after 2 days incubation at 37 ° C. To more closely mimic the conditions of the mutation assay 108 filter cells (NR5280) were included with the test cell. The filler cells are unable to grow under these conditions by virtue of a non-revertible autotrophic marker (cysG::
TnlO). Assay for DNA methylation Whole cell or plasrnid D N A was isolated by a rapid isolation procedure (Davis et al., 1980) and digested with Sau3AI, MboI and DpnI (New England Biolabs, Inc., Beverly, MA). D N A was
134
electrophoresed in 0.8% agarose gel, stained with ethidium bromide and photographed.
TABLE 2
Mutagenicity assays Mutation assays were carried out according to the published plate-test procedures (Ames et al., 1975). A small volume of an aqueous stock solution of 9AA or ICR191 (Polysciences Inc., Warrington, PA) (no greater than 100 /~l/p!ate ) and 100 /xl of fresh stationary-phase bacterial culture grown in NYA medium (ca. 2 x 108 cells) were added to 2.0 ml of overlay agar held at 46 o C. The contents of the tubes were mixed and poured over the surface of agar plates containing 25 ml of minimal E selection medium supplemented with 20 # g / m l L-tryptophan and 0.8 # g / m l L-histidine. Overlay agar was allowed to solidify and the plates were incubated at 37 ° C for 3 days. At the end of the incubation period back-mutation yields were scored at least in duplicate with the aid of an Artek 880 automatic counter (Artek Systems Corp., Farmingdale, NY). All experiments were repeated 3 times. The doses of 9AA and ICR 191 used in these assays were shown to have no effect on cell survival.
Other conditions
Assays for TnlO activity For measurement of Tnl0-mediated rearrangements extending into the gal region, cells (approximately 2 x 105) incorporated in 2 ml of overlay agar were poured onto supplemented minimal E medium and incubated overnight for aproximately 15 h. Plates were overlayed with a mixture of 2 ml of 2 x overlay agar and 1.5 ml of 20% D-galactose, and i n c u b a t e d for 2 days. Galactose-resistant colonies were counted. To test for retention of TnlO, colonies were purified by streaking twice for single colonies on supplemented minimal E medium containing 1.2% Dgalactose, and tested for tetracycline resistance by streaking on N Y A plates containing 25 /xg/ml tetracycline. For all strains 65-75% of colonies were tetracycline sensitive. Results
(1) Isolation and characterization of NR5076. The mutant NR5076 was isolated after treatment of N R 5038 (uvr-304, trpES, hisC3076) with
SENSITIVITY TO 2-AMINOPURINE
m u t H l O 1 :: T n 5 b pMQ148 dam + b Methionine c
Zone diameter a (mm) dam +
dam- 1
d a m -2
10 9 10 12
38 9 9 37
21 9 10 12.5
a Diameters are averages of 3 determinations. b m u t H l O 1 : : T n 5 o r p M Q 1 4 8 d a m + w a s i n t r o d u c e d i n t o the s t r a i n (see M e t h o d s ) . c M e t h i o n i n e (45 # g / m l ) w a s a d d e d to the plates.
N-methyl-N'-nitro-N'nitrosoguanidine, and detected in a screen for mutants with reduced levels of reversion of the hisC3076 frameshift marker by 9-aminoacridine. Mutants isolated in this way were screened for sensitivity to the lethal effects of several agents including 2-aminopurine. As 2AP sensitivity is characteristic of dam- mutants in E. coli (Glickman et al., 1978), and the 2AP sensitivity of dam- mutants is reversed by the presence of mutations in the genes of the mismatch repair pathway (McGraw and Marinus, 1980; Glickman and Radman, 1980), the mutHlOl::Tn5 allele was introduced into all mutants showing 2AP sensitivity. For 2 mutants (NR5265 and NR5076) 2AP sensitivity was reversed by the mutH allele (Table 2). NR5265 dam-1 has been shown to be completely devoid of D N A adenine methylation, and the mutation (dam-l) maps in the D N A adenine methylase gene (Ritchie et al., 1986). NR5076 showed a level of 2AP sensitivity intermediate between that of NR5038 (dam +) and of NR5265 (dam-l) (see Table 2). Introduction of the plasmid pMQ148 which contains a functional E. coli dam ÷ gene also reversed the 2AP sensitivity of NR5076. As pMQ148 carries only the functional E. coli D N A adenine methylase gene and reverses the phenotypic characteristics of dammutants in E. coli (Arraj and Marinus, 1983) and S. typhimurium (Ritchie et al., 1986), this result suggests that the mutation in NR5076 is located in the dam gene. The 2AP sensitivity was cotransducible with cysG (44.5% cotransduction) which confirms the location of the mutation (dam-2) in
135 the D N A adenine methylase gene (Marinus, 1973; Ritchie et al., 1986). An interesting feature of the mutant NR5076 (dam-2) is the reversal of 2AP sensitivity by addition of methionine to the medium (see Table 2). This effect is not observed in strains carrying the dam-1 mutation, nor is the level of 2AP sensitivity of dam ÷ strains reduced by methionine. The dam-2 mutation was introduced into a non-mutagenized background, and this strain NR5308 was used in all further experiments.
(2) DNA adenine methylation in NR5308 The methylation status of adenine residues in the sequence - G A T C - was investigated using the restriction enzymes Sau3A, MboI and DpnI, all of which recognize this sequence. Sau3A cleaves both methylated and unmethylated sites, MboI cleaves only unmethylated sites and DpnI cleaves only methylated sites. Cellular D N A was isolated from NR5308 (dam-2) and digested with the restriction enzymes (Sau3A, DpnI and MboI). The D N A was completely digested by Sau3A and by DpnI and not detectably digested by MboI (not shown). These results suggest that the majority of - G A T C - sites are methylated in NR5308 and thus that the dam-2 mutation does not lead to substantial inactivation of the D N A adenine methylase. To obtain a more sensitive assay for the presence of unmethylated sites in D N A from NR5308, the plasmid pBR322 was introduced and plasmid D N A isolated from an unamplified culture. This D N A was completely digested by Sau3A and DpnI (not shown). Some nicking and cleavage by MboI was observed in plasmid D N A isolated from both NR5068 (dam ÷) and NR5308 (dam-), suggesting the presence of a single unmethylated or hemimethylated site in a small number of molecules, but no difference between the strains was detectable. Thus the dam-2 mutation does not lead to a detectable reduction in the level of methylation of - G A T C - sequences in the DNA. (3) Spontaneous mutability An elevated spontaneous mutation frequency is characteristic of dam- mutants (Bale et al., 1979; Glickman, 1979). Spontaneous reversion of the
100
100
~o
survival
200
9AA (ugl01ate) 30O 40O 500
600
,o
[
Dam + d,.am-2
1
Fig, 1.9-Aminoacridinesensitivityof dam÷, dare-1 and dam-2 strains. 9-Aminoacridinewas included with 500 cells and filler cells in overlay agar. A, NR5068 dam÷;@,NR5293 darn-l; I, NR5308 dam-2. Values are averages of 3 independent duplicate determinations.
base substitution marker trpE8 and the frameshift marker hisC3076 in the dam-2 strain was not significantly different (21.3 Trp ÷ and 5.9 His + colonies/plate) from the dam ÷ strain (21.0 Trp ÷ and 5.8 His + colonies/plate). Spontaneous reversion of these markers is elevated 5- and 20-fold respectively in the dam-1 strain (Ritchie et al., 1986).
(4) 9-Aminoacridine sensitioity The procedure used to isolate the dam-1 and dam-2 strain involved screening for reduced numbers of 9AA-induced His + revertants of the hisC3076 mutation. In the case of the dam-1 strain we have reported that 9AA mutagenesis is in fact dramatically increased (Ritchie et al., 1986). The detection of this strain may have resulted from the increased sensitivity of the dam-1 strain to the lethal effects of 9AA (Fig. 1). Sensitivity of dam-1 strains to 9AA is reversed by introduction of the mutHl05 :: T n 5 mutation or the dam + plasmid pMQ148 (not shown). However, the dam-2 strain is not significantly sensitive to 9AA (Fig. 1).
136
IT
1500
|
/ / HIs' 1000
/
l /
o.m+
dam-1
7
dam
C')dam-I/ / dam-,2/ DM--"Q"~4 ~ / DMQ148/ -Dam+/
/
/
colonies per plate 500
50
loo
9AA (pg/plate)
150
200
250
Fig. 2. 9-Aminoacridine-induced reversion of hisC3076 in dam +, dam.1 and dam-2 strains with and without the plasmid pMQ148 dam +. 9-Aminoacridine was included with 2 - 5 × 1 0 ~ cells in 2 ml of overlay agar on minimal plates with limiting histidine. A, NR5068, O, NR5293 dam-l; I , NR5308 dam-2; A, NR5068 dam+/pMQ148; o, NR5293 dam-1/pMQ148; D, NR5308 dam-2~ pMQ148. Values are averages of 3 independent duplicate determinations.
(5) Frameshift mutagenesis
The dose-response curves for 9AA-induced reversion of the hisC3076 marker in the dam-2 and dam ÷ strains, using plates assays, are shown in Fig. 2. The dose response for the dam-1 strain is also shown for comparison. The 2-component dose-response curve observed in dam + strains presumably results from efficient repair 9AA-induced errors up to a threshold dose beyond which the capacity of the mismatch repair system to correct errors is exceeded and mutations are induced. In the dam-2 strain the threshold dose is increased approximately 2-fold indicating a greater capacity for error correction in this strain. Addition of methionine to the medium does not reverse this effect (not shown). Reduced susceptibility of the dam-2 strain to the mutagenic effects of 9AA was also observed when the dose response to 9AA is assayed following treatment of a log-phase culture with various doses of 9AA for 30 min. The number of His ÷ revertants induced per #g 9AA was 10.2 for NR5038 (dam +) and 2.6 for NR5076 (dam-2). The dose-response curves for dam +, dam-1 and dam-2 strains carrying the dam + plasmid pMQ148 are also shown in Fig. 2. All 3 plasmidcontaining strains exhibit similar dose-response
curves, and the threshold dose for mutation induction is about 2-fold lower than for the dam ÷ strain without the plasrnid. This result implies a reduced capacity for mismatch repair in the presence of the multicopy dam ÷ plasmid.
2000I | 15001
/
,~ Dam*/pMO148 laam-~21pM0148 //
'1' ° ' " '
I I fo..._,/,Mo,,, III
I / I /
""
::°":oooI # //
'"'"
5
0
o lO
f/ 0
///
..-°Lm:
~
40 60 80 100 ICR191(pg/plale)
150
Fig. 3. ICR191-induced reversion of hisC3076 in dam +, dam-I and dam-2 strains with and without the plasmid pMQ148 dam +. Strains used are described in the legend to Fig. 2. Values are averages of 3 independent duplicate determinations.
137
The dose-response curves for ICR191-induced reversion of hisC3076 in darn +, dam-1 and dam-2 strains with and without pMQ148 are shown in Fig. 3. The dose response with ICR191 in the darn + strain differs from that observed with 9AA, and is essentially linear, though a small threshold dose effect is observed. However, mismatch repair also plays a significant role in avoidance of ICR191-induced errors as evidenced by the dramatic increase in slope observed for the darn-1 strain. In contrast to the results obtained for 9AA, the darn-2 mutation has little or no effect on induction of frameshift errors by ICR191. ICR191 binds covalently to DNA, forming lesions which can be excised by the enzymes of the uorABC pathway (Newton et al., 1972; Imray and MacPhee, 1976). However in the uvr-304 background, the darn + and darn-2 strains also show identical dose responses (not shown), though substantially lower doses are required for equivalent levels of mutagenesis. Thus removal of mutagenic lesions by the uvrABC pathway is not responsible for the failure to detect a difference between the dam + and darn-2 strains when ICR191 is used as the mutagen. Introduction of the multicopy plasmid pMQ148 darn + reverses the effect of the darn-1 mutation and leads to a 2-fold increase in the slope for both darn ÷ and darn-2 strains (Fig. 3). Thus it appears that the dam-2 mutation has no effect on the repair of frameshift errors induced by ICR191, but that overproduction of the E. coli DNA adenine methylase by pMQ148 reduces the amount of error correction. (6) Transposon activity
Mutants deficient in DNA adenine methylase also exhibit increased T n l O and Tn5 transposon activity (Arraj and Marinus, 1983; Lundblad and Kleckner, 1984; Roberts et al., 1985). For T n l O this effect has been shown to result from the influence of the methylation status of particular - G A T C - sites on the expression of transposition functions (Roberts et al., 1985). Tnl0-promoted deletions and rearrangements were assayed by induction of galactose resistance in strains carrying galE and a closely situated zbi-812 :: T n l O insertion (Table 3). Tnl0-promoted deletions and rearrangements which extend into the gal operon
TA B LE 3
TnlO A C T I V I T Y IN dam +, darn-1 A N D dam-2 S T R A I N S Allele
Tn I 0-promoted rearrangements gale zbi-812 :: TnlO G a l r c o l o n i e s / p l a t e a
+
77 715 82
dam-1 darn-2
a Values are averages of 3 independent determinations. The numbers of cells per plate, before selection for G a l r was applied, was identical for the 3 strains.
reverse the sensitivity to galactose conferred by the gale mutation. No difference was observed between the darn + and darn-2 strains, while a 10-fold increase was observed in the darn-1 strain. The increased T n l O activity in the darn-1 strain is consistent with observations in E. coli d a m strains. Discussion The dam-2 mutant described here differs in several respects from the DNA adenine methylation-deficient (darn-) mutants which have been previously described. Most strikingly the DNA of the dam-2 mutant appears to be fully methylated indicating that the methylase is not grossly defective in methylation activity. The many phenotypic effects of darn- mutations have been reviewed by Marinus (1984). For several of these phenotypes (increased spontaneous mutability, increased Tn 10 activity, increased sensitivity to 9AA), the darn-2 strain resembles the wild-type dam + strain. In contrast, dam-2 resembles darn-1 in conferring sensitivity to 2AP and in that this sensitivity is reversed by a mutation in one of the genes (mutH) of the mismatch repair pathway. However the 2AP sensitivity of the dam-2 strain is lower than that of the dam-1 strain and is reversed by addition of methionine to the assay medium. Finally the susceptibility to mutagenesis by 9AA is reduced 2-fold by dam-2 but increased substantially by darn-1. The proposal that DNA adenine methylation provides the mismatch repair system with a mechanism for strand discrimination is now well supported by experiments using in vitro constructed heteroduplexes of bacteriophage h and M13 DNA
138 which contain a single mismatch (Dohet et al., 1985; Kramer et al., 1984; Pukkila et al., 1983; Radman et al., 1985) or single base additions or deletions (Dohet et al., 1986; Radman et al., 1985). When only 1 strand of the heteroduplex is methylated, the unmethylated strand is preferentially repaired. When neither strand is methylated, repair occurs without strand discrimination, and when both strands are methylated no repair is observed. For the E. coli chromosome, mismatch repair is believed to be a post-replicative process which rectifies errors generated during replication and not corrected by proofreading functions. It has been proposed that the newly synthesized strand is transiently unmethylated at - G A T C - sites in the D N A at the replication fork and that these hemimethylated sites provide the strand discrimination mechanism. This notion is supported by evidence that the newly synthesized strand is undermethylated (Marinus, 1976; Lyons and Schendel, 1984). Furthermore, the spontaneous mutation frequency is substantially increased in d a m - mutants of E. coli (Bale et al., 1979; Glickman, 1979) and in S. typhimurium (Ritchie et al., 1986) as well as in mismatch repair-deficient mutL, H, S and uorD mutants (Rydberg, 1978; Shanabruch et al., 1981; Glickman and Radman, 1980) and the spectrum of spontaneous mutations is similar in d a m - and rout- strains (Glickman, 1979; Leong et al., 1986). Comparison of the dose-response curves for 9AA-induced frameshift reversion events in d a m and d a m - strains of E. coli (Mohn et al., 1984) and S. typhimurium (Ritchie et al., 1986) indicate that in dam ÷ strains the majority of 9AA-induced frameshifts errors are accurately and efficiently repaired by the adenine methylation-instructed mismatch repair pathway. As mutation induction occurs at low 9AA doses in the d a m - strain, the virtual absence of induced revertants in the dam ÷ strain at 9AA doses below 100 ~tg 9 A A / p l a t e can be attributed to complete repair of induced lesions. At higher doses the capacity of the repair pathways becomes saturated and mutation induction is observed. As methylation-instructed repair is primarily a post-replicative process, this observation also implies that the majority of 9AA-induced errors occur during the replication process. The increased levels of 9AA required to induce
frameshift errors in the dam-2 strain described here can most simply be explained by postulating that in dam-2 the methylation of hemimethylated - G A T C - sites at the replication fork occurs more slowly than in the dam ÷ strain, allowing a longer period in which the mismatch repair system can discriminate between strands and preferentially repair the newly synthesized strand. A similar explanation can account for the reduced levels of 9AA required for mutagenesis in dam ÷ and darn-2 strains carrying the multicopy pMQ148 dam ÷ plasmid. Overproduction of the D N A adenine methylase should increase the rate of methylation at the form and thus reduce the amount of mismatch repair which can occur. Indeed it has been shown that the spontaneous mutation frequency of dam ÷ strains is elevated by introduction of dam ÷ plasmids which overproduce the D N A adenine methylase 20-50-fold (Herman and Modrich, 1981) or 300-500-fold (Marinus et al., 1984). However a plasmid which results in 15-fold over-expression is without effect (Marinus et al., 1984). It is possible that for spontaneous mutations the number of replication errors is sufficiently low that the rate of methylation does not become rate limiting until very high levels of over-expression are achieved. Moreover the basal level of methylase activity may vary considerably in different strains. We have found that the spontaneous reversion of the frameshift marker hisC3076 and the base substitution marker trpE8 is unaffected in the dam-2 strain and also unaffected by the presence of pMQ148 plasmid which includes the dam gene in a pBR322 vector. However, using 9AA-induced mutagenesis as an assay could be expected to be far more sensitive to small variations in the rate of methylation as many more mismatch-correctable lesions are present in this case. When ICR191 was used as the mutagen several differences from 9AA are observed. The dose response does not exhibit the clear threshold, seen with 9AA. Nonetheless, the dramatic increase in slope observed with the dam-1 strain indicates that mismatch repair is responsible for correction of the majority of ICR191-induced errors. Though the multicopy dam ÷ plasmid increases the slope of the dose-response curve, no difference between the dam + and dam-2 strains is observed. We are
139 unable to offer a simple explanation for these findings. The proposal that methylation at the fork is slower in the dam-2 strain fails to explain the 2AP-sensitive phenotype of the dam-2 strain. To date the 2AP sensitivity of d a m - strains has been explained by proposing that because the D N A is unmethylated, strand discrimination cannot occur and breaks are introduced into both strands by the enzymes of the mut pathway in response to the presence of 2AP in the DNA. Sensitivity then reflects the introduction of double-strand breaks. The presence of nicks in the D N A is supported by the fact that 2AP induces the SOS response, d a m but not in dam + strains (Bebenek and Janion, 1985; Craig et al., 1984), and the involvement of the mismatch repair pathway is supported by the finding that mutH, mutL and mutS mutations reverse both the 2AP sensitivity (Glickman and Radman, 1980; McGraw and Marinus, 1980) and the 2AP SOS-inducing ability (Craig et al., 1984; Bebenek and Janion, 1985) of d a m - strains. More direct evidence for mismatch repair-induced double-strand breaks in unmethylated D N A has recently been reported (Doutriaux et al., 1986; Wang and Smith, 1986). However, an indication that this explanation for sensitivity of d a m - strains to base analogues is somewhat simplistic comes from studies on the adenine analogues 2-amino-N6-hydroxyadenine and 2-amino-N6-methoxyadenine. Mutagenesis by these analogues is elevated in d a m - strains, but d a m - strains are sensitive only to the lethal effects of 2-amino-N6-hydroxy adenine (Bebenek and Janion, 1983). Furthermore, both analogues resemble 2AP in that they induce the SOS response in d a m - cells, but not in d a m - r o u t - cells (Bebenek and Janion, 1985). In the case of the dam-2 mutant described here, the D N A is essentially fully methylated. Introduction of nicks in both strands leading to doublestrand breaks cannot be invoked to explain the 2AP sensitivity, as the parental strand is methylated and nicks should only be introduced into the newly synthesized unmethylated strand. However the mismatch repair pathway appears to be involved, as a dam-2 m u t H : : T n 5 strain is no longer 2AP sensitive. Furthermore, the D N A adenine methylase is also involved as the presence of the E. coli dam + methylase gene on a multicopy
plasmid also reverse 2AP sensitivity. A possible explanation is that the dam-2 methylase is particularly sensitive to the cellular level of its substrate methyl donor, S-adenosylmethionine (SAM), and that 2AP affects the adenine nucleotide pools, thus reducing the SAM concentration and leading to extensive demethylation of D N A , 2AP-induced cell death would then occur by a mechanism akin to that which occurs in d a m - cells. If addition of methionine restores the SAM levels to normal, this would account for the reversal of the 2AP sensitivity of the dam-2, but not the dam-1 strain, by addition of methionine to the medium. The S. typhirnurium dam-1 strain exhibits sensitivity of 9AA, and this effect is also reversed by introduction of a m u t H mutation or the dam + plasmid. However the mechanism of 9AA sensitivity cannot be identical to that for 2AP sensitivity, as the dam-2 strain is not significantly more sensitive than the wild type. The introduction of nicks on either strand at the site of 9AA-induced errors is in this case a plausible explanation for 9AA sensitivity in the dam-1 strain. However, 9AA does not appear to induce the SOS response in d a m - E. coli cells (Quillardet and Hofnung, personal communication). Further analysis of the dam-2 mutant is clearly required to clarify some of the issues raised in this study. In particular direct measurement of the degree of methylation at the fork, or a study of the properties of the purified methylase is required to determine if the rate of D N A adenine methylation at the replication fork is reduced in this mutant.
Acknowledgements We thank Trish Hayes for competent technical assistance.
References Ames, B.N., and H.J. Whitfield Jr. (1966) Frameshift mutagenesis in Salmonella, Cold Spring Harbor Symp. Quant. Biol., 31, 221-225. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsometest, Mutation (Res., 31, 347-364. Arraj, J.A., and M.G. Marinus (1983) Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam + gene, J. Bacteriol., 153, 562-565.
140 Bale, A., M. d'Alarcao and M.G. Marinus (1979) Characterization of DNA adenine methylation mutants of Escherichia coil K12, Mutation Res., 59, 157-165. Bebenek, K., and C. Janion (1983) Involvement of the mismatch repair system in base analogne-induced mutagenesis, Mol. Gen. Genet., 1991, 276-281. Bebenek, K., and C. Janion (1985) Ability of base analogues to induce the SOS response: effect of a dam mutation and mismatch repair system, Mol. Gen. Genet., 201, 519-524. Calos, M.P., and J.H. Miller (1981) Genetic and sequence analysis of frameshift mutations induced by ICR191, J. Mol. Biol., 153, 39-66. Craig, R.J., J.A. Arraj and M.G. Marinus (1984) Induction of damage inducible (SOS) repair in dam mutants of Escherichia coil exposed to 2-aminopurine, Mol. Gen. Genet., 194, 539-540. Davis, R.W., D. Botstein and J.R. Roth (1980) A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Dohet, C., R. Wagner and M. Radman (1985) Repair of defined single base-pair mismatches in Escherichia coli, Proc. Natl. Acad. Sci. (U.S.A.), 82, 503-505. Dohet, C., R. Wagner and M. Radman (1986) Methyl-directed repair of frame,shift mutations in heteroduplex DNA, Proc. Natl. Acad. Sci. (U.S.A.), 83, 3395-3397. Doutriaux, M-P., R. Wagner and M. Radman (1986) Mismatch stimulated killing, Proc. Natl. Acad. Sci. (U.S.A.), 83, 2576-2578. Glickrnan, B.W. (1979) Spontaneous mutagenesis in Escherichia coil strains lacking 6-methyladenine residues in their D N A , Mutation Res., 61, 153-162. Glickman, B.W., and M. Radman (1980) Escherichia coil mutator mutants deficient in rnethylation-instructed D N A mismatch correction, Proc. Natl. Acad. SCI. (U.S.A.), 77, 1063-1067. Glickman, B.W., P. van den Elsen and M. Radman (1978) Induced mutagenesis in dam- mutants of Escherichia coil, A role of 6-methyladenine residues in mutation avoidance, Mol. Gen. Genet., 163, 307-312. Herman, G.E., and P. Modrich (1981) Escherichia coli K-12 clones that overproduce dam methylase are hypermutable, J. Bacteriol., 145, 64,*-646. Imray, P., and D.G. MacPhec (1976) Spontaneous and induced mutability of frameshift strains of Salmonella typhimurium carrying uvrB and poL4 mutations, Mutation Res., 34, 35-42. Kramer, B., W. Kramer and H.-J. Fritz (1984) Different b a s e / base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coil, Cell, 38, 879-887. Lederberg, E.M., and S.N. Cohen (1974) Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid, J. Bacteriol., 119, 1072-1074. Leong, P.-M., H.C. Hsia and J.H. Miller (1986) Analysis of spontaneous base substitutions generated in mismatch-repair-deficient strains of Escherichia coil, J. Bacteriol., 168, 412-416. Lundblad, V., and N. Kleckner (1984) Mismatch repair muta-
tions of Escherichia coli K12 enhance transposon excision, Genetics, 109, 3-19. Lyons, S.M., and P.F. Schendel (1984) Kinetics of methylation in Escheriehia coli K-12, J. Bacteriol., 159, 421-423. McGraw, B.R., and M.G. Marinus (1980) Isolation and characterization of Dam + revertants and suppression mutants that modify secondary phenotypes of dam-3 strains of Escherichia coli K12, Mol. Gen. Genet., 178, 309-315. Marinus, M.G. (1973) Location of methylation genes on the Escheriehia coli K-12 genetic map, Mol. Gen. Genet., 127, 47-55. Marinus, M.G. (1976) Adenine methylation of Okazaki fragments in Eseherichm coli, J. Bacteriol., 128, 853-854. Marinus, M.G. (1984) Methylation of procaryotic DNA, in: A. Razin, H. Cedar and A. Riggs (Eds.), DNA Methylation, Biochemistry and Biological Significance, Springer, New York, pp. 81-109. Marinus, M.G., A. Poteete and J.A. Arraj (1984) Correlation of DNA adenine methylase activity with spontaneous mutability in Escherichia coli K12, Gene, 28, 123-125. Mohn, G.R., P.R.M. Kerklaan, A.A. van Zeeland, J. Ellenberger, R.A. Baan, P.H.M. Lohman and F.W. Pons (1984) Methodologies for the determination of various genetic effects in permeable strains of E. coli K-12 differing in DNA repair capacity, Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal mediated assays, Mutation Res., 125, 153-184. Newton, A., D. Masys, E. Leonardi and D. Wygal (1972) Association of induced frameshift mutagenesis and DNA replication in Escherichia coli, Nature (London), New Biol., 236, 19-22. Ohta, T., N. Nakamura, M. Moriya, Y. Shirasu and T. Kada (1984) The SOS function-inducing activity of chemical mutagens in Escherichia coil, Mutation Res., 131,101-109. Podger, D.M., and R.M. Hall (1985) Induction of the SOS response by ICR191 does not influence frameshift mutagenesis at the hisC3076 marker of Salmonella typhimurium, Mutation Res., 142, 87-91. Pukkila, P.J., J. Peterson, G. Herman, P. Modrich and M. Meselson (1983) Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coil, Genetics, 104, 571-582. Radman, M., C. Dohet, M. Jones, M.P. Doutriaux, F. Laengle-Roualt, G. Maenhaut-Michel and R. Wagner (1985) Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli, Biochemie, 67, 745-752. Ripley, L.S., and A. Clark (1986) Frameshift mutations produced by proflavine in bacteriophage T4: specificity within a hotspot, Proc. Natl. Acad. Sci. (U.S.A.), 83, 6954-6958. Ritchie, L.J., R.M. Hall and D.M. Podger (1986) A mutant Salmonella typhimurium LT2 deficient in DNA adenine methylation, J. Bacteriol., 167, 420-422. Roberts, D., B.C. Hoopes, W.R. McLure and N. Kleckner (1985) IS10 transposition is regulated by DNA adenine methylation, Cell, 43, 117-130. Roth, J.R. (1974) Frameshift mutations, Annu. Rev. Genet., 8, 319-346.
141
Rydberg, B. (1978) Bromouracil mutagenesis and mismatch repair in mutation strains of Escherichia coli, Mutation Res., 52, 11-24. Shanabruch, W.G., I. Behlau and C.G. Walker (1981) Spontaneous Salmonella typhimurium LT2 generated by insertion of transposable elements, J. Bacteriol., 147, 827-835. Skopek, T.R., and F. Hutchinson (1984) Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191, Mol. Gen. Genet., 195, 418-423.
Vogel, H.J., and D.M. Bonner (1956) Acetyiornithase of Escherichia coli: partial purification and some properties, J. Biol. Chem., 218, 97-106. Wang, T.-C.V., and K.C. Smith (1986) Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair, J. Bacteriol., 165, 1023-1025.