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Roles of the mutagenesis proteins SamA′B and MucA′B in chemically induced frameshift mutagenesis in Salmonella typhimurium hisD3052
Mutation Research 398 Ž1998. 33–42
Roles of the mutagenesis proteins SamAX B and MucAX B in chemically induced frameshift mutagenesis in Salmonella typhimurium hisD3052 Petr Gruz, Keiko Matsui, Toshio Sofuni, Takehiko Nohmi
)
DiÕision of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan Received 25 June 1997; revised 2 October 1997; accepted 9 October 1997
Abstract The mutagenesis induced by ultraviolet light and many chemicals in Escherichia coli is largely dependent upon the proteins encoded by the umuDC operon and their analogs. In Salmonella typhimurium, there are two sets of umuDC-like operons: the umuDCST operon in the chromosome and the samAB operon located on the 60-MDa cryptic plasmid. The former operon, but not the latter, confers UV mutability on S. typhimurium. Nevertheless, the samAB operon, when carried on high-copy-number plasmids, can efficiently promote UV mutagenesis. In order to characterize the function of samAB in greater detail, we have compared the abilities of MucAX B and a putative activated form of SamAB, i.e. SamAX B, to promote chemically induced frameshift mutagenesis in S. typhimurium hisD3052. MucAX B is an activated form of the products of mucAB, which is the most potent umuDC analog characterized so far. We have used four plasmids, each carrying samAX , samB, mucAX or mucB with a lac promoter instead of their own promoters. The results indicated that under the conditions of elevated expression, SamAX B can promote chemically induced frameshift mutagenesis by furylfuramide, aflatoxin B1, 1-nitropyrene, and 1,8-dinitropyrene, with efficiencies comparable to, or even better than, MucAX B. Increase of the levels of expression enhanced the ability of SamAX B to promote the mutagenesis, while it decreased that of MucAX B. Surprisingly, the elevated expression of MucB alone significantly enhanced the frameshift mutagenesis induced by 1-nitropyrene and 1,8-dinitropyrene, whereas the elevated expression of SamB, MucAX and SamAX did not enhance it. These results suggest that the abilities of SamAX B and MucAX B to promote mutagenesis strongly depend on their levels of expression. The possible roles of these mutagenesis proteins in chemically induced frameshift mutagenesis are discussed. q 1998 Elsevier Science B.V. Keywords: samAB; umuDC; mucAB; Frameshift mutagenesis; Mutagenesis protein; Salmonella typhimurium
1. Introduction In Escherichia coli, mutagenesis by ultraviolet light ŽUV. and most chemicals requires the expres) Corresponding author. Tel. q81 Ž3. 3700-9873; fax: q81 Ž3. 3707-6950; E-mail [email protected]
sion of the umuDC operon w1,2x. The umuDC operon is located at about 26 min on the E. coli chromosome and encodes the 15.1- and 47.7-kDa proteins UmuD and UmuC, respectively w3–5x. Expression of the umuDC operon and other homologous operons, such as mucAB, impAB, samAB and rumAB, is regulated as a part of SOS response of E. coli, in
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 7 - 6
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P. Gruz et al.r Mutation Research 398 (1998) 33–42
which an activated form of RecA mediates the cleavage of repressor protein LexA w5–10x. The activated form of RecA also mediates the cleavage of UmuD w11–13x. The resulting carboxyterminal fragment of UmuD, UmuDX , is necessary and sufficient for the role of UmuD in UV mutagenesis w12x. UmuDX forms a homodimer that makes a complex with UmuC w14x. The appearance of the UmuDX C protein complex switches DNA repair from homologous recombination to SOS mutagenesis w15x. Although the exact mechanism is not known, evidence suggests that the complex, UmuDX –UmuDX –UmuC, and RecA are involved in translesion DNA synthesis, in which DNA polymerases incorporate an incorrect nucleotide opposite a non-instructional or mis-instructive lesion and then continue DNA synthesis w16–18x. The products of umuDC and its analogs are also involved in frameshift mutagenesis induced by chemicals. In studies of N-2-acetylaminofluorene ŽAAF., umuDC promotes translesion DNA synthesis in repetitive sequences having AAF adducts, and mucAB contributes to the generation of y2 frameshift mutations within alternating GC sequences w19,20x. In the lacZ mutagenesis assays in E. coli, mucAB efficiently enhances frameshifts induced by nitracrine, 4-nitroquinoline-N-oxide Ž4NQO ., 2-Ž2-furyl.-3-Ž5-nitro-2-furyl.-acrylamide Žfurylfuramide., mitomycin C ŽMMC. or N-methylN X-nitro-N-nitrosoguanidine ŽMNNG. w21,22x. Induced reversions of the hisD3052 frameshift allele in Salmonella typhimurium are also strongly influenced by mucAB and umuDC w23,24x. In the cases of benzow axpyrene, aflatoxin B 1 ŽAFB1., and 1-nitropyrene Ž1-NP., more frameshift mutations are observed if the mucAX B or umuDX C genes are expressed, instead of mucAB or umuDC w25,26x. The mucAX B and umuDX C genes encode the activated forms of MucAB and UmuDC, respectively w5,27x. The umuDCST operon, which is located on the chromosome of S. typhimurium, enhances y2 frameshift mutations induced by 1-nitropyrene and 1,8-dinitropyrene within alternating GC sequences in the hisD3052 allele w24x. There are, however, mutagens which seem to induce frameshift mutations efficiently in the absence of any umuDC analog. Examples include 2-nitrofluorene Ž2-NF., 2-amino-3-methyldipyridow1,2-a:3X ,2X-dximidazole ŽGlu-P-1., and 2amino-3-methylimidazoŽ4,5-f .quinoline ŽIQ. w24,28x.
In S. typhimurium, there is another umuDC-like operon, i.e. samAB, carried on a 60-MDa cryptic plasmid, which is commonly present in derivatives of S. typhimurium strain LT2 w9,29–31x. The samA gene product shares the putative cleavage site as well as the amino acids involved in the mechanism of the cleavage reaction conserved in other UmuD-like proteins, and so the SamA protein is probably also activated by proteolysis for its role in mutagenesis w9x. The SamAX B proteins are fully active in UV mutagenesis w32x. In order to further characterize the functions of samAB, we have compared the abilities of SamAX B and MucAX B to promote chemically induced frameshift mutagenesis in S. typhimurium D3052. Using four plasmids each carrying samAX , samB, mucAX , or mucB under the control of an inducible promoter, we also examined the effects of levels of expression and the expression of single mutagenesis proteins on promoting frameshift mutagenesis w32x. The effects of levels of expression was examined because our previous work suggests that the ability of samAB to promote UV mutagenesis strongly depends on the levels of expression w31x. Based on our results, we discuss the roles of these proteins in chemically induced frameshift mutagenesis in S. typhimurium.
2. Materials and methods 2.1. Bacterial strains and plasmids The bacterial strains used in this study were derivatives of the strain S. typhimurium YG5147 Ž h isD 3 0 5 2 , D u Õ rB , D u m u D C S T ::K m R , D samAB::CmR . ŽTable 1.. The plasmids expressing different mutagenesis proteins were pYG8524 Ž mucAX ., pYG8529 Ž samAX ., pYG8517 Ž mucB . and pYG8518 Ž samB . and the control plasmids were pET-16b and pYG8516. The details of the plasmid constructions and their transfer to the strain YG5147 were described previously w32x. 2.2. Chemicals and mutagenesis assay The sources of chemicals used in this study are as follows: furylfuramide, Glu-P-1 and isopropyl b-Dthiogalactopyranoside ŽIPTG. from Wako Pure
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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Table 1 Bacterial strains and plasmids Strain or plasmid S. typhimurium TA1538 YG5147 Plasmid pET-16b pYG8524 pYG8529 pYG8516 pYG8517 pYG8518
Description
Reference
hisD3052 gal DŽ chl, uÕrB, bio ., rfa As TA1538 but is D umuDCST ::KmR , D samAB::CmR
w25x w24x
Expression vector with T7lac promoter ŽpBR322 derivative. X MucA expression vector with lac promoter ŽpBR322 derivative. X SamA expression vector with lac promoter ŽpBR322 derivative. pSC101 derivative with lac promoter MucB expression vector with lac promoter ŽpSC101 derivative. SamB expression vector with lac promoter ŽpSC101 derivative.
Novagen w32x w32x w32x w32x w32x
Chemical Industries, Osaka, Japan; 1-NP and 2-NF from Tokyo Kasei Kogyo, Tokyo, Japan; AFB1 from Sigma Chemicals, St. Louis, MO; 1,8-dinitropyrene Ž1,8-DNP. was a gift from Dr. Naoki Miyata, National Institute of Health Sciences, Tokyo, Japan. Rat liver 9000 = g supernatant fraction ŽS9. from animals pretreated with phenobarbital and 5,6-benzoflavone was purchased from Kikkoman, Chiba, Japan. The standard mutagenesis assay ŽAmes test. was carried out employing the optional 20-min preincubation procedure w33x. Overnight cultures were grown in Bacto Nutrient Broth supplemented with 0.2% glucose and the following antibiotics, as required: kanamycin at 10 mgrml q chloramphenicol at 5
mgrml Žfor all YG5147 based strains., ampicillin at 50 mgrml Žfor strains harboring plasmids pYG8524, pYG8529 or pET-16b. and tetracycline at 10 mgrml Žfor strains harboring plasmids pYG8517, pYG8518 or pYG8516.. The experiments were repeated at least twice and two plates were used for each dose. The mean values of Hisq revertants per plate are indicated in Tables 2 and 3, and Figs. 1 and 2. 3. Results In order to compare the abilities of SamAX B and MucAX B to promote frameshift mutagenesis in S. typhimurium, we have used a set of plasmids, each
Table 2 Chemically induced frameshift mutations promoted by the elevated levels of expression of various combinations of mutagenesis proteins Mutagenesis protein
Induced Hisq revertantsrplate Furylfuramide Ž0.01 mgrplate.
Homologous combinations X SamA plus SamB 78 X MucA plus MucB 9 Heterologous combinations X SamA plus MucB 4 X MucA plus SamB 7 Vectors alone 0
AFB1 Ž0.1 mgrplate.
1-NP Ž0.25 mgrplate.
1,8-DNP Ž2.5 ngrplate.
2-NF Ž5 mgrplate.
Glu-P-1 Ž25 ngrplate.
337 471
300 160
2466 1615
1003 839
978 244
195 178 22
87 63 0
1510 2147 252
907 1240 747
994 883 969
The following plasmids were introduced into a strain S. typhimurium YG5147 for the expression of mutagenesis proteins: pYG8529 and X X X pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and pYG8517 for SamA plus MucB; pYG8524 X and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. The mutagenicity tests were conducted with more than four doses of the chemicals and the mean numbers of induced Hisq revertantsrplate at the indicated doses are presented. The numbers of induced Hisq revertantsrplate were calculated by subtracting the numbers of spontaneous Hisq revertantsrplate from those of Hisq revertantsrplate of treated groups. The doses used for the presentation of the results were selected within the linear range of the dose–response curves. The highest doses examined for each chemical are as follows: furylfuramide, 0.05 mgrplate; aflatoxin B1 , 0.1 mgrplate; 1-nitropyrene, 0.5 mgrplate; 1,8-dinitropyrene, 10 ngrplate; 2-nitrofluorene, 10 mgrplate; Glu-P-1, 50 ngrplate. The dose–response curves of aflatoxin B1 , 1,8-dinitropyrene and 2-nitrofluorene are presented in Fig. 1.
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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Table 3 X X Effects of IPTG induction on the chemically induced mutagenesis promoted by SamA B and MucA B Mutagenesis protein
X
SamA q SamB X MucA q MucB
Ratio of the numbers of induced Hisq revertantsrplate in the presence and the absence of IPTG Furylfuramide Ž0.01 mgrplate. ŽyIPTG.
AFB1 Ž0.1 mgrplate. ŽyIPTG.
1-NP Ž0.25 mgrplate. ŽyIPTG.
1,8-DNP Ž2.5 ngrplate. ŽyIPTG.
2-NF Ž5 mgrplate. ŽyIPTG.
Glu-P-1 Ž25 ngrplate. ŽyIPTG.
1.2 Ž64. 0.1 Ž94.
1.5 Ž224. 0.6 Ž799.
1.7 Ž176. 0.5 Ž355.
1.7 Ž1481. 0.8 Ž2019.
1.0 Ž1038. 0.6 Ž1451.
1.1 Ž857. 0.5 Ž490.
The ratio was calculated by dividing the numbers of induced Hisq revertantsrplate with IPTG by those without IPTG. The numbers in X X parentheses indicate the mean numbers of induced Hisq revertants of the strain expressing SamA B or MucA B by the chemicals at the indicated dosesrplate in the absence of IPTG. The numbers in the presence of IPTG are presented in Table 2.
carrying samAX , samB, mucAX , or mucB w32x. For each of plasmids, the entire SOS regulatory region plus the promoter was replaced with the lac promoter, and the coding regions of samA and mucA
were changed to those of samAX and mucAX . The plasmids carrying samAX and mucAX are derivatives of pBR322, while those carrying samB and mucB are derivatives of pSC101, which is compatible with
Fig. 1. Dose–response curves of aflatoxin B1 , 1,8-dinitropyrene and 2-nitrofluorene with a strain S. typhimurium YG5147 harboring two X X X plasmids expressing A-proteins ŽSamA or MucA . and B-proteins ŽSamB or MucB.. The following plasmids were introduced into a strain X X S. typhimurium YG5147: pYG8529 and pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and X X pYG8517 for SamA plus MucB; pYG8524 and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. IPTG Ž0.2 mM. X X was added to the top agar to induce the expression of the mutagenesis proteins. v, vectors alone; `, MucA q MucB; ', SamA q SamB; X X ^, MucA q SamB; B, SamA q MucB.
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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X X Fig. 2. The effects of expression of single proteins ŽMucA , SamA , MucB or SamB. on the mutability of a strain S. typhimurium YG5147 by 1-nitropyrene and 1,8-dinitropyrene. The following plasmids were introduced into a strain S. typhimurium YG5147: pYG8524 for X X MucA ; pYG8529 for SamA ; pYG8517 for MucB; pYG8518 for SamB. Control is a strain YG5147 without plasmids. IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. The values represent the mean numbers of Hisq revertants per plate of two plates. The height of bars represents the mean number of induced Hisq revertants per plate.
the pBR322 replicon. The low-copy-number of plasmid pSC101 assures lower expression of B-genes, i.e. samB and mucB genes, relative to AX genes, i.e. the samAX and mucAX genes, which resembles the physiological situation. Thus, either simultaneous expression of any combination of AX plus B genes, or single expression of any gene, is possible with these plasmids. In addition, the expression of each genes is SOS-independent and can be modulated by IPTG. We have introduced the plasmids into S. typhimurium YG5147, which lacks both umuDCST and samAB, and bears the hisD3052 allele, the reversion of which is sensitive to frameshift mutagens w24x. We examined the mutagenicities of 6 compounds, i.e. furylfuramide, AFB1, 1-NP, 1,8-DNP, 2-NF and Glu-P-1, in the strains harboring these plasmids, in the presence of IPTG. In some cases, we also examined the mutagenicities in the absence of IPTG. Based on the results, we classified the 6 mutagens into three groups, as follows. Group 1 includes furylfuramide and AFB1. The characteristic of this group is that the strains harboring the plasmids carrying any combination of the AX plus B genes exhibited higher mutabilities to the
chemicals than those harboring vector plasmids alone ŽTable 2.. In particular, cells expressing homologous combinations of the mutagenesis proteins, i.e. SamAX plus SamB or MucAX plus MucB, appeared to be more mutable than those expressing the heterologous combinations, i.e. SamAX plus MucB or MucAX plus SamB. This is typically shown in the mutagenicity of AFB1. Apparent efficiencies of the combinations of the mutagenesis proteins were: MucAX plus MucB ) SamAX plus SamB ) SamAX plus MucBs MucAX plus SamB ) no mutagenesis proteins ŽFig. 1.. In the case of furylfuramide, no mutagenicity was detected with the strains harboring the vector plasmids alone. Thus, it is clear that the elevated expression of SamAX B can promote frameshift mutagenesis with efficiencies comparable to or even better than that of MucAX B ŽTable 2.. In the absence of IPTG treatment, SamAX B could promote frameshift mutagenesis with efficiencies 30–70% of those of MucAX B ŽTable 3.. Group 2 includes 1-NP and 1,8-DNP. As with group 1, the strains harboring the plasmids carrying any combination of AX plus B genes exhibited higher mutabilities than those harboring vector plasmids
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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Table 4 Spontaneous frameshift mutations promoted by the expression of various combinations of mutagenesis proteins Induced Hisq revertantsrplate
y IPTG qIPTG
X
X
X
X
Vectors alone
SamA q SamB
MucA q MucB
SamA q MucB
MucA q SamB
20 " 9 17 " 10
21 " 9 22 " 5
66 " 20 49 " 15
20 " 8 24 " 6
20 " 11 24 " 13
The following plasmids were introduced into a strain S. typhimurium YG5147 for the expression of mutagenesis proteins: pYG8529 and X X X pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and pYG8517 for SamA plus MucB; pYG8524 X and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. qIPTG means that IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. yIPTG means that no IPTG was added. The values are means standard deviations of 6 independent cultures with two plates Ž n s 12..
alone ŽTable 2.. However, 1-NP and 1,8-DNP are distinct from group 1, in that significant induced mutagenesis was observed when MucB protein alone was expressed in the presence of IPTG ŽFig. 2.. Other proteins, such as SamB, MucAX or SamAX , did not affect mutagenesis induced by nitropyrenes, in the presence of IPTG. In the cases of furylfuramide, AFB1, 2-NF and Glu-P-1, we did not observe any significant enhancement of mutagenesis even when MucB was expressed in the presence of IPTG. Results for 1,8-DNP were different from those for 1-NP in the following two respects: first, the strain expressing the heterologous combinations of the mutagenesis proteins, i.e. SamAX plus MucB or MucAX plus SamB, exhibited similar mutabilities with 1,8DNP, but not 1-NP, to those expressing the homologous combinations, i.e. SamAX plus SamB or MucAX plus MucB ŽFig. 1, Table 2.; second, in the absence of mutagenesis proteins, i.e. in the control strain harboring the vector plasmids alone, a low, but significant, mutagenicity was detected with 1,8-DNP, but not with 1-NP ŽTable 2.. Group 3 includes 2-NF and Glu-P-1. We could not observe any enhancing effect of the plasmids on the promotion of mutagenesis induced by these compounds ŽTable 2 and Fig. 1.. In fact, in the case of Glu-P-1, increase expression of MucAX B reduced the mutagenesis. Negative effects were also observed in the absence of IPTG treatments ŽTable 3.. For spontaneous reversion of the hisD3052 allele, only the combination of MucAX plus MucB enhanced the numbers of spontaneous Hisq revertants per plate for strain YG5147 ŽTable 4.. The extent of enhancement was about 3-fold when comparing the numbers of spontaneous Hisq revertants per plate for
strain YG5147 harboring the plasmids expressing MucAX plus MucB, and the strain harboring the vector plasmids alone. This is in contrast with the strong enhancement of spontaneous base substitution mutagenesis by MucAX B: the combination of MucAX plus MucB produced more than 500 spontaneous Hisq revertants per plate in the strain YG5144 carrying hisG428 w32x.
4. Discussion The allele hisD3052 of S. typhimurium is reverted mainly by y2 frameshift mutations occurring at the CGCGCGCG hot spot w34x and is therefore used for exploring the y2 frameshift mutagenesis pathway w24x. We have introduced plasmids carrying samAX , samB, mucAX , or mucB under control by a lac promoter w32x into the strain YG5147, which carries the hisD3052, and examined the mutagenicities of several chemicals. The strain YG5147 lacks both umuDCST and samAB, which simplifies interpretation of the results w24x. Strikingly, the compounds tested could be classified into three groups. Frameshift mutagenesis induced by the chemicals of group 1 Žfurylfuramide and AFB1. and 1-NP was strongly dependent on the expression of mutagenesis proteins ŽTable 2 and Fig. 1.. In the absence of mutagenesis proteins, furylfuramide and 1-NP exhibited no significant mutagenicity in the doses we examined. The homologous expression of mutagenesis proteins, i.e. SamAX plus SamB or MucAX plus MucB, more efficiently promoted the mutagenesis by these chemicals than the heterologous expression, i.e. SamAX plus MucB or MucAX plus SamB. It
P. Gruz et al.r Mutation Research 398 (1998) 33–42
seems that these compounds resemble UV light, in that the formation of homologous combinations of SamAX plus SamB or MucAX plus MucB is strongly required for their maximum mutagenesis. It has been reported that abasic sites arise from the DNA modifications by furylfuramide w35x and AFB1 w36x. Abasic sites are DNA lesions that strongly block DNA replication w18x. Furthermore, N-Ždeoxyguanosin-8yl.-1-aminopyrene, a major DNA adduct formed by reductively activated 1-NP, also represents a strong replication block w37,38x. Hence, the chemicals whose DNA lesions strongly hinder DNA replication may require authentic pairs of mutagenesis proteins such as SamAX B or MucAX B for the maximum frameshift mutagenesis. In the case of 1,8-DNP, all of the combinations of the tested AX plus B proteins enhanced mutagenesis with similar efficiencies ŽTable 2 and Fig. 1.. This is markedly different from the chemical frameshift mutagenesis or UV-induced base-substitution mutagenesis. 1,8-DNP very efficiently induces y2 frameshift deletions at the repetitive CG sequence in hisD3052 of S. typhimurium w24x. It has been known that more than 95% of the total DNA adducts formed by the treatment of 1-nitroso-8-nitropyrene, an activated form of 1,8-DNP, in E. coli are N-Ž2X-deoxyguanosin-8-yl.-1-amino-8-nitropyrene w39x. Thus, it seems plausible that, unlike pyrimidine dimers or abasic sites, the DNA adduct induced by 1,8-DNP does not severely block DNA replication when it is formed at the CG repetitive sequence. 1,8-DNP exhibited low, but significant, mutagenicity in the strain YG5147 harboring vector plasmids alone, suggesting that DNA polymerases can bypass the mutagenic DNA lesion at the CG repetitive sequence, to some extent, even in the absence of mutagenesis proteins. The results obtained with 2-NF and Glu-P-1 Žgroup 3. suggest that these compounds induce mutations without the assistance of the mutagenesis proteins ŽTable 2 and Fig. 1.. This conclusion is in accordance with the report that the mutagenicities of 2-NF and Glu-P-1 in S. typhimurium D3052 are not affected by the deletion of umuDCST genes w24x. It is likely that the DNA adducts generated by 2-NF and Glu-P-1 do not strongly block replication and the DNA polymerase itself can easily bypass these structures by a slippage mechanism. It is indeed reported that the DNA polymerase III holoenzyme replicates
39
DNA template containing N-Ždeoxyguanosin-C8-yl.2-aminofluorene, which is thought to be formed from the metabolically activated 2-NF, with high efficiency w40x. This DNA adduct is also formed by 2-aminofluorene Ž2-AF ., following oxidative metabolism. Recently, it was reported that the products of umuDC are not required for frameshift mutagenesis by IQ. IQ–DNA adducts behave more similarly to 2-NF or 2-AF adducts than acetylaminofluorene ŽAAF. adducts, which strongly hinder DNA replication w28x. Since the expression of the mutagenesis proteins in our plasmids is controllable by IPTG w32x, we examined the effects of their levels of expression on the mutagenesis. The expression level should be similar between SamAX and MucAX and between SamB and MucB because the expression from all the AX and B genes is controlled by the identical Shine– Dalgarno sequence linked to the identical lac promoter. Interestingly, IPTG treatments exhibited opposite effects on the mutabilities of the strains expressing SamAX B or MucAX B. In strains harboring the plasmids expressing SamAX B, IPTG treatments increased the mutabilities of the host strains to furylfuramide, AFB1, 1-NP and 1,8-DNP ŽTable 3.. This is consistent with the previous knowledge that samAB on multi-copy-number plasmids more efficiently promotes UV mutagenesis than that on low-copy-number plasmids w9,31x. UmuC-like proteins are very unstable and tend to aggregate w14,41x. SamB may be less stable than MucB, and thus a higher level of expression of SamAX B is necessary to carry out the extent of mutagenesis promoted by a lower level of expression of MucAX B. In contrast, in the strains harboring the plasmids expressing MucAX B, IPTG treatments reduced the mutabilities of the host strains for all the chemicals tested ŽTable 3.. In particular, IPTG treatment substantially reduced the mutability by furylfuramide of the strains expressing MucAX B to furylfuramide ŽTables 2 and 3.. Interestingly, it is reported that expression of excess UmuC results in a non-mutable phenotype of E. coli w42x. Mutagenesis proteins, such as SamAX B, MucAX B or UmuDX C, could have optimum intracellular concentrations for mutagenesis, and they might rather decrease the mutagenesis if they are expressed too much. It could be possible, however, that this apparent reduction of mutagenesis by the elevated expression of MucAX B
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P. Gruz et al.r Mutation Research 398 (1998) 33–42
is due to the killing of host cells rather than the real reduction of mutagenesis because it has been known that the overexpression of UmuDC is associated with cell lethality w43x. It is also reported that overexpression of UmuDX C results in the in vitro formation of UmuDX C–ssDNA complex, which blocks DNA replication w41x. A surprising observation was that the elevated expression of MucB alone could promote the frameshift mutagenesis induced by 1-NP and 1,8DNP ŽFig. 2.. It is conceivable that DNA polymerases associated with MucB, without the assistance of MucAX or SamAX , can bypass the lesions induced by nitropyrenes. It is proposed that RecA targets the MucAX or UmuDX to DNA lesions, thereby allowing the complexes of MucAX B or UmuDX C to interact with DNA polymerases at the lesions w16x. MucAX and UmuDX proteins interact with RecA as well as MucB and UmuC, respectively w44x. Thus, MucB protein will not be well targeted to DNA lesions if MucAX is absent. The results shown in Fig. 2 raise the possibility that a trace amount of MucB protein alone is sufficient for supporting translesion bypass of DNA polymerases at the lesions induced by nitropyrenes. It could also be interesting to examine whether the MucB protein requires the presence of RecA for such AX-independent enhancement of nitropyrene-induced mutagenesis. There is also another conceivable explanation for this AX-independent enhancement. The chromosome or 60-MDa cryptic plasmid of S. typhimurium might encode some yet undiscovered UmuD-like protein that is able to target the MucB protein to the nitropyrene-modified DNA. For instance, UmuDX-like protein termed HumD was found in the genome of bacteriophage P1 w45x and an UmuC-like protein termed DinPrB has been recently cloned from the genome of E. coli w46x. Such Umu-like proteins might interact with mutagenesis proteins and modulate their activity under certain circumstances. The expression of SamAX B or MucAX B did not significantly increase the number of spontaneous Hisq revertants of the hisD3052 allele ŽTable 4.. This is in contrast with the previous results that the expression of MucAX B markedly increases the number of spontaneous Hisq base-substitution revertants of the hisG428 or hisG46 alleles w32x. Spontaneous frameshift mutations mainly occur by slippage of
DNA replication at repetitive DNA sequences w47x. S. typhimurium hisD3052 allele has a CGCGCGCG sequence, which is a hot spot for spontaneous as well as chemically induced frameshift mutations w24x. Thus, we suggested that neither SamAX B nor MucAX B efficiently promotes the slippage error of DNA replication at the repetitive sequences. Alternatively, the spontaneous mutations might occur at repetitive sequences having endogenous DNA lesions that do not strongly hinder DNA replication as in the case of chemicals of group 3 in this study w48x. In summary, we have demonstrated that the elevated expression of SamAX B can promote frameshift mutagenesis by furylfuramide, AFB1, 1-NP and 1,8DNP with efficiencies comparable to or even higher than that of MucAX B. The abilities of SamAX B and MucAX B to promote mutagenesis strongly depended on the levels of their expression. We propose that B-proteins rather than AX-proteins play a crucial role in mutagenesis because the elevated expression of MucB alone could promote frameshift mutagenesis by 1-NP and 1,8-DNP.
Acknowledgements We thank Dr. P.D. Josephy, University of Guelph, for critically reading the manuscript. This work was supported by grants-in-aid for Scientific Research on Priority Areas Ž08280104. from the Ministry of Education, Science, Sports and Culture of Japan, Science and Technology Agency of Japan, and the Japanese Health Science Foundation. P.G. was supported by fellowships from the Japanese Health Science Foundation and the Science and Technology Agency.
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Roles of the mutagenesis proteins SamAX B and MucAX B in chemically induced frameshift mutagenesis in Salmonella typhimurium hisD3052 Petr Gruz, Keiko Matsui, Toshio Sofuni, Takehiko Nohmi
)
DiÕision of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158, Japan Received 25 June 1997; revised 2 October 1997; accepted 9 October 1997
Abstract The mutagenesis induced by ultraviolet light and many chemicals in Escherichia coli is largely dependent upon the proteins encoded by the umuDC operon and their analogs. In Salmonella typhimurium, there are two sets of umuDC-like operons: the umuDCST operon in the chromosome and the samAB operon located on the 60-MDa cryptic plasmid. The former operon, but not the latter, confers UV mutability on S. typhimurium. Nevertheless, the samAB operon, when carried on high-copy-number plasmids, can efficiently promote UV mutagenesis. In order to characterize the function of samAB in greater detail, we have compared the abilities of MucAX B and a putative activated form of SamAB, i.e. SamAX B, to promote chemically induced frameshift mutagenesis in S. typhimurium hisD3052. MucAX B is an activated form of the products of mucAB, which is the most potent umuDC analog characterized so far. We have used four plasmids, each carrying samAX , samB, mucAX or mucB with a lac promoter instead of their own promoters. The results indicated that under the conditions of elevated expression, SamAX B can promote chemically induced frameshift mutagenesis by furylfuramide, aflatoxin B1, 1-nitropyrene, and 1,8-dinitropyrene, with efficiencies comparable to, or even better than, MucAX B. Increase of the levels of expression enhanced the ability of SamAX B to promote the mutagenesis, while it decreased that of MucAX B. Surprisingly, the elevated expression of MucB alone significantly enhanced the frameshift mutagenesis induced by 1-nitropyrene and 1,8-dinitropyrene, whereas the elevated expression of SamB, MucAX and SamAX did not enhance it. These results suggest that the abilities of SamAX B and MucAX B to promote mutagenesis strongly depend on their levels of expression. The possible roles of these mutagenesis proteins in chemically induced frameshift mutagenesis are discussed. q 1998 Elsevier Science B.V. Keywords: samAB; umuDC; mucAB; Frameshift mutagenesis; Mutagenesis protein; Salmonella typhimurium
1. Introduction In Escherichia coli, mutagenesis by ultraviolet light ŽUV. and most chemicals requires the expres) Corresponding author. Tel. q81 Ž3. 3700-9873; fax: q81 Ž3. 3707-6950; E-mail [email protected]
sion of the umuDC operon w1,2x. The umuDC operon is located at about 26 min on the E. coli chromosome and encodes the 15.1- and 47.7-kDa proteins UmuD and UmuC, respectively w3–5x. Expression of the umuDC operon and other homologous operons, such as mucAB, impAB, samAB and rumAB, is regulated as a part of SOS response of E. coli, in
0027-5107r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 7 . 0 0 2 3 7 - 6
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P. Gruz et al.r Mutation Research 398 (1998) 33–42
which an activated form of RecA mediates the cleavage of repressor protein LexA w5–10x. The activated form of RecA also mediates the cleavage of UmuD w11–13x. The resulting carboxyterminal fragment of UmuD, UmuDX , is necessary and sufficient for the role of UmuD in UV mutagenesis w12x. UmuDX forms a homodimer that makes a complex with UmuC w14x. The appearance of the UmuDX C protein complex switches DNA repair from homologous recombination to SOS mutagenesis w15x. Although the exact mechanism is not known, evidence suggests that the complex, UmuDX –UmuDX –UmuC, and RecA are involved in translesion DNA synthesis, in which DNA polymerases incorporate an incorrect nucleotide opposite a non-instructional or mis-instructive lesion and then continue DNA synthesis w16–18x. The products of umuDC and its analogs are also involved in frameshift mutagenesis induced by chemicals. In studies of N-2-acetylaminofluorene ŽAAF., umuDC promotes translesion DNA synthesis in repetitive sequences having AAF adducts, and mucAB contributes to the generation of y2 frameshift mutations within alternating GC sequences w19,20x. In the lacZ mutagenesis assays in E. coli, mucAB efficiently enhances frameshifts induced by nitracrine, 4-nitroquinoline-N-oxide Ž4NQO ., 2-Ž2-furyl.-3-Ž5-nitro-2-furyl.-acrylamide Žfurylfuramide., mitomycin C ŽMMC. or N-methylN X-nitro-N-nitrosoguanidine ŽMNNG. w21,22x. Induced reversions of the hisD3052 frameshift allele in Salmonella typhimurium are also strongly influenced by mucAB and umuDC w23,24x. In the cases of benzow axpyrene, aflatoxin B 1 ŽAFB1., and 1-nitropyrene Ž1-NP., more frameshift mutations are observed if the mucAX B or umuDX C genes are expressed, instead of mucAB or umuDC w25,26x. The mucAX B and umuDX C genes encode the activated forms of MucAB and UmuDC, respectively w5,27x. The umuDCST operon, which is located on the chromosome of S. typhimurium, enhances y2 frameshift mutations induced by 1-nitropyrene and 1,8-dinitropyrene within alternating GC sequences in the hisD3052 allele w24x. There are, however, mutagens which seem to induce frameshift mutations efficiently in the absence of any umuDC analog. Examples include 2-nitrofluorene Ž2-NF., 2-amino-3-methyldipyridow1,2-a:3X ,2X-dximidazole ŽGlu-P-1., and 2amino-3-methylimidazoŽ4,5-f .quinoline ŽIQ. w24,28x.
In S. typhimurium, there is another umuDC-like operon, i.e. samAB, carried on a 60-MDa cryptic plasmid, which is commonly present in derivatives of S. typhimurium strain LT2 w9,29–31x. The samA gene product shares the putative cleavage site as well as the amino acids involved in the mechanism of the cleavage reaction conserved in other UmuD-like proteins, and so the SamA protein is probably also activated by proteolysis for its role in mutagenesis w9x. The SamAX B proteins are fully active in UV mutagenesis w32x. In order to further characterize the functions of samAB, we have compared the abilities of SamAX B and MucAX B to promote chemically induced frameshift mutagenesis in S. typhimurium D3052. Using four plasmids each carrying samAX , samB, mucAX , or mucB under the control of an inducible promoter, we also examined the effects of levels of expression and the expression of single mutagenesis proteins on promoting frameshift mutagenesis w32x. The effects of levels of expression was examined because our previous work suggests that the ability of samAB to promote UV mutagenesis strongly depends on the levels of expression w31x. Based on our results, we discuss the roles of these proteins in chemically induced frameshift mutagenesis in S. typhimurium.
2. Materials and methods 2.1. Bacterial strains and plasmids The bacterial strains used in this study were derivatives of the strain S. typhimurium YG5147 Ž h isD 3 0 5 2 , D u Õ rB , D u m u D C S T ::K m R , D samAB::CmR . ŽTable 1.. The plasmids expressing different mutagenesis proteins were pYG8524 Ž mucAX ., pYG8529 Ž samAX ., pYG8517 Ž mucB . and pYG8518 Ž samB . and the control plasmids were pET-16b and pYG8516. The details of the plasmid constructions and their transfer to the strain YG5147 were described previously w32x. 2.2. Chemicals and mutagenesis assay The sources of chemicals used in this study are as follows: furylfuramide, Glu-P-1 and isopropyl b-Dthiogalactopyranoside ŽIPTG. from Wako Pure
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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Table 1 Bacterial strains and plasmids Strain or plasmid S. typhimurium TA1538 YG5147 Plasmid pET-16b pYG8524 pYG8529 pYG8516 pYG8517 pYG8518
Description
Reference
hisD3052 gal DŽ chl, uÕrB, bio ., rfa As TA1538 but is D umuDCST ::KmR , D samAB::CmR
w25x w24x
Expression vector with T7lac promoter ŽpBR322 derivative. X MucA expression vector with lac promoter ŽpBR322 derivative. X SamA expression vector with lac promoter ŽpBR322 derivative. pSC101 derivative with lac promoter MucB expression vector with lac promoter ŽpSC101 derivative. SamB expression vector with lac promoter ŽpSC101 derivative.
Novagen w32x w32x w32x w32x w32x
Chemical Industries, Osaka, Japan; 1-NP and 2-NF from Tokyo Kasei Kogyo, Tokyo, Japan; AFB1 from Sigma Chemicals, St. Louis, MO; 1,8-dinitropyrene Ž1,8-DNP. was a gift from Dr. Naoki Miyata, National Institute of Health Sciences, Tokyo, Japan. Rat liver 9000 = g supernatant fraction ŽS9. from animals pretreated with phenobarbital and 5,6-benzoflavone was purchased from Kikkoman, Chiba, Japan. The standard mutagenesis assay ŽAmes test. was carried out employing the optional 20-min preincubation procedure w33x. Overnight cultures were grown in Bacto Nutrient Broth supplemented with 0.2% glucose and the following antibiotics, as required: kanamycin at 10 mgrml q chloramphenicol at 5
mgrml Žfor all YG5147 based strains., ampicillin at 50 mgrml Žfor strains harboring plasmids pYG8524, pYG8529 or pET-16b. and tetracycline at 10 mgrml Žfor strains harboring plasmids pYG8517, pYG8518 or pYG8516.. The experiments were repeated at least twice and two plates were used for each dose. The mean values of Hisq revertants per plate are indicated in Tables 2 and 3, and Figs. 1 and 2. 3. Results In order to compare the abilities of SamAX B and MucAX B to promote frameshift mutagenesis in S. typhimurium, we have used a set of plasmids, each
Table 2 Chemically induced frameshift mutations promoted by the elevated levels of expression of various combinations of mutagenesis proteins Mutagenesis protein
Induced Hisq revertantsrplate Furylfuramide Ž0.01 mgrplate.
Homologous combinations X SamA plus SamB 78 X MucA plus MucB 9 Heterologous combinations X SamA plus MucB 4 X MucA plus SamB 7 Vectors alone 0
AFB1 Ž0.1 mgrplate.
1-NP Ž0.25 mgrplate.
1,8-DNP Ž2.5 ngrplate.
2-NF Ž5 mgrplate.
Glu-P-1 Ž25 ngrplate.
337 471
300 160
2466 1615
1003 839
978 244
195 178 22
87 63 0
1510 2147 252
907 1240 747
994 883 969
The following plasmids were introduced into a strain S. typhimurium YG5147 for the expression of mutagenesis proteins: pYG8529 and X X X pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and pYG8517 for SamA plus MucB; pYG8524 X and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. The mutagenicity tests were conducted with more than four doses of the chemicals and the mean numbers of induced Hisq revertantsrplate at the indicated doses are presented. The numbers of induced Hisq revertantsrplate were calculated by subtracting the numbers of spontaneous Hisq revertantsrplate from those of Hisq revertantsrplate of treated groups. The doses used for the presentation of the results were selected within the linear range of the dose–response curves. The highest doses examined for each chemical are as follows: furylfuramide, 0.05 mgrplate; aflatoxin B1 , 0.1 mgrplate; 1-nitropyrene, 0.5 mgrplate; 1,8-dinitropyrene, 10 ngrplate; 2-nitrofluorene, 10 mgrplate; Glu-P-1, 50 ngrplate. The dose–response curves of aflatoxin B1 , 1,8-dinitropyrene and 2-nitrofluorene are presented in Fig. 1.
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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Table 3 X X Effects of IPTG induction on the chemically induced mutagenesis promoted by SamA B and MucA B Mutagenesis protein
X
SamA q SamB X MucA q MucB
Ratio of the numbers of induced Hisq revertantsrplate in the presence and the absence of IPTG Furylfuramide Ž0.01 mgrplate. ŽyIPTG.
AFB1 Ž0.1 mgrplate. ŽyIPTG.
1-NP Ž0.25 mgrplate. ŽyIPTG.
1,8-DNP Ž2.5 ngrplate. ŽyIPTG.
2-NF Ž5 mgrplate. ŽyIPTG.
Glu-P-1 Ž25 ngrplate. ŽyIPTG.
1.2 Ž64. 0.1 Ž94.
1.5 Ž224. 0.6 Ž799.
1.7 Ž176. 0.5 Ž355.
1.7 Ž1481. 0.8 Ž2019.
1.0 Ž1038. 0.6 Ž1451.
1.1 Ž857. 0.5 Ž490.
The ratio was calculated by dividing the numbers of induced Hisq revertantsrplate with IPTG by those without IPTG. The numbers in X X parentheses indicate the mean numbers of induced Hisq revertants of the strain expressing SamA B or MucA B by the chemicals at the indicated dosesrplate in the absence of IPTG. The numbers in the presence of IPTG are presented in Table 2.
carrying samAX , samB, mucAX , or mucB w32x. For each of plasmids, the entire SOS regulatory region plus the promoter was replaced with the lac promoter, and the coding regions of samA and mucA
were changed to those of samAX and mucAX . The plasmids carrying samAX and mucAX are derivatives of pBR322, while those carrying samB and mucB are derivatives of pSC101, which is compatible with
Fig. 1. Dose–response curves of aflatoxin B1 , 1,8-dinitropyrene and 2-nitrofluorene with a strain S. typhimurium YG5147 harboring two X X X plasmids expressing A-proteins ŽSamA or MucA . and B-proteins ŽSamB or MucB.. The following plasmids were introduced into a strain X X S. typhimurium YG5147: pYG8529 and pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and X X pYG8517 for SamA plus MucB; pYG8524 and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. IPTG Ž0.2 mM. X X was added to the top agar to induce the expression of the mutagenesis proteins. v, vectors alone; `, MucA q MucB; ', SamA q SamB; X X ^, MucA q SamB; B, SamA q MucB.
P. Gruz et al.r Mutation Research 398 (1998) 33–42
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X X Fig. 2. The effects of expression of single proteins ŽMucA , SamA , MucB or SamB. on the mutability of a strain S. typhimurium YG5147 by 1-nitropyrene and 1,8-dinitropyrene. The following plasmids were introduced into a strain S. typhimurium YG5147: pYG8524 for X X MucA ; pYG8529 for SamA ; pYG8517 for MucB; pYG8518 for SamB. Control is a strain YG5147 without plasmids. IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. The values represent the mean numbers of Hisq revertants per plate of two plates. The height of bars represents the mean number of induced Hisq revertants per plate.
the pBR322 replicon. The low-copy-number of plasmid pSC101 assures lower expression of B-genes, i.e. samB and mucB genes, relative to AX genes, i.e. the samAX and mucAX genes, which resembles the physiological situation. Thus, either simultaneous expression of any combination of AX plus B genes, or single expression of any gene, is possible with these plasmids. In addition, the expression of each genes is SOS-independent and can be modulated by IPTG. We have introduced the plasmids into S. typhimurium YG5147, which lacks both umuDCST and samAB, and bears the hisD3052 allele, the reversion of which is sensitive to frameshift mutagens w24x. We examined the mutagenicities of 6 compounds, i.e. furylfuramide, AFB1, 1-NP, 1,8-DNP, 2-NF and Glu-P-1, in the strains harboring these plasmids, in the presence of IPTG. In some cases, we also examined the mutagenicities in the absence of IPTG. Based on the results, we classified the 6 mutagens into three groups, as follows. Group 1 includes furylfuramide and AFB1. The characteristic of this group is that the strains harboring the plasmids carrying any combination of the AX plus B genes exhibited higher mutabilities to the
chemicals than those harboring vector plasmids alone ŽTable 2.. In particular, cells expressing homologous combinations of the mutagenesis proteins, i.e. SamAX plus SamB or MucAX plus MucB, appeared to be more mutable than those expressing the heterologous combinations, i.e. SamAX plus MucB or MucAX plus SamB. This is typically shown in the mutagenicity of AFB1. Apparent efficiencies of the combinations of the mutagenesis proteins were: MucAX plus MucB ) SamAX plus SamB ) SamAX plus MucBs MucAX plus SamB ) no mutagenesis proteins ŽFig. 1.. In the case of furylfuramide, no mutagenicity was detected with the strains harboring the vector plasmids alone. Thus, it is clear that the elevated expression of SamAX B can promote frameshift mutagenesis with efficiencies comparable to or even better than that of MucAX B ŽTable 2.. In the absence of IPTG treatment, SamAX B could promote frameshift mutagenesis with efficiencies 30–70% of those of MucAX B ŽTable 3.. Group 2 includes 1-NP and 1,8-DNP. As with group 1, the strains harboring the plasmids carrying any combination of AX plus B genes exhibited higher mutabilities than those harboring vector plasmids
P. Gruz et al.r Mutation Research 398 (1998) 33–42
38
Table 4 Spontaneous frameshift mutations promoted by the expression of various combinations of mutagenesis proteins Induced Hisq revertantsrplate
y IPTG qIPTG
X
X
X
X
Vectors alone
SamA q SamB
MucA q MucB
SamA q MucB
MucA q SamB
20 " 9 17 " 10
21 " 9 22 " 5
66 " 20 49 " 15
20 " 8 24 " 6
20 " 11 24 " 13
The following plasmids were introduced into a strain S. typhimurium YG5147 for the expression of mutagenesis proteins: pYG8529 and X X X pYG8518 for SamA plus SamB; pYG8524 and pYG8517 for MucA plus MucB; pYG8529 and pYG8517 for SamA plus MucB; pYG8524 X and pYG8518 for MucA plus SamB; pET-16b and pYG8516 for vectors alone. qIPTG means that IPTG Ž0.2 mM. was added to the top agar to induce the expression of the mutagenesis proteins. yIPTG means that no IPTG was added. The values are means standard deviations of 6 independent cultures with two plates Ž n s 12..
alone ŽTable 2.. However, 1-NP and 1,8-DNP are distinct from group 1, in that significant induced mutagenesis was observed when MucB protein alone was expressed in the presence of IPTG ŽFig. 2.. Other proteins, such as SamB, MucAX or SamAX , did not affect mutagenesis induced by nitropyrenes, in the presence of IPTG. In the cases of furylfuramide, AFB1, 2-NF and Glu-P-1, we did not observe any significant enhancement of mutagenesis even when MucB was expressed in the presence of IPTG. Results for 1,8-DNP were different from those for 1-NP in the following two respects: first, the strain expressing the heterologous combinations of the mutagenesis proteins, i.e. SamAX plus MucB or MucAX plus SamB, exhibited similar mutabilities with 1,8DNP, but not 1-NP, to those expressing the homologous combinations, i.e. SamAX plus SamB or MucAX plus MucB ŽFig. 1, Table 2.; second, in the absence of mutagenesis proteins, i.e. in the control strain harboring the vector plasmids alone, a low, but significant, mutagenicity was detected with 1,8-DNP, but not with 1-NP ŽTable 2.. Group 3 includes 2-NF and Glu-P-1. We could not observe any enhancing effect of the plasmids on the promotion of mutagenesis induced by these compounds ŽTable 2 and Fig. 1.. In fact, in the case of Glu-P-1, increase expression of MucAX B reduced the mutagenesis. Negative effects were also observed in the absence of IPTG treatments ŽTable 3.. For spontaneous reversion of the hisD3052 allele, only the combination of MucAX plus MucB enhanced the numbers of spontaneous Hisq revertants per plate for strain YG5147 ŽTable 4.. The extent of enhancement was about 3-fold when comparing the numbers of spontaneous Hisq revertants per plate for
strain YG5147 harboring the plasmids expressing MucAX plus MucB, and the strain harboring the vector plasmids alone. This is in contrast with the strong enhancement of spontaneous base substitution mutagenesis by MucAX B: the combination of MucAX plus MucB produced more than 500 spontaneous Hisq revertants per plate in the strain YG5144 carrying hisG428 w32x.
4. Discussion The allele hisD3052 of S. typhimurium is reverted mainly by y2 frameshift mutations occurring at the CGCGCGCG hot spot w34x and is therefore used for exploring the y2 frameshift mutagenesis pathway w24x. We have introduced plasmids carrying samAX , samB, mucAX , or mucB under control by a lac promoter w32x into the strain YG5147, which carries the hisD3052, and examined the mutagenicities of several chemicals. The strain YG5147 lacks both umuDCST and samAB, which simplifies interpretation of the results w24x. Strikingly, the compounds tested could be classified into three groups. Frameshift mutagenesis induced by the chemicals of group 1 Žfurylfuramide and AFB1. and 1-NP was strongly dependent on the expression of mutagenesis proteins ŽTable 2 and Fig. 1.. In the absence of mutagenesis proteins, furylfuramide and 1-NP exhibited no significant mutagenicity in the doses we examined. The homologous expression of mutagenesis proteins, i.e. SamAX plus SamB or MucAX plus MucB, more efficiently promoted the mutagenesis by these chemicals than the heterologous expression, i.e. SamAX plus MucB or MucAX plus SamB. It
P. Gruz et al.r Mutation Research 398 (1998) 33–42
seems that these compounds resemble UV light, in that the formation of homologous combinations of SamAX plus SamB or MucAX plus MucB is strongly required for their maximum mutagenesis. It has been reported that abasic sites arise from the DNA modifications by furylfuramide w35x and AFB1 w36x. Abasic sites are DNA lesions that strongly block DNA replication w18x. Furthermore, N-Ždeoxyguanosin-8yl.-1-aminopyrene, a major DNA adduct formed by reductively activated 1-NP, also represents a strong replication block w37,38x. Hence, the chemicals whose DNA lesions strongly hinder DNA replication may require authentic pairs of mutagenesis proteins such as SamAX B or MucAX B for the maximum frameshift mutagenesis. In the case of 1,8-DNP, all of the combinations of the tested AX plus B proteins enhanced mutagenesis with similar efficiencies ŽTable 2 and Fig. 1.. This is markedly different from the chemical frameshift mutagenesis or UV-induced base-substitution mutagenesis. 1,8-DNP very efficiently induces y2 frameshift deletions at the repetitive CG sequence in hisD3052 of S. typhimurium w24x. It has been known that more than 95% of the total DNA adducts formed by the treatment of 1-nitroso-8-nitropyrene, an activated form of 1,8-DNP, in E. coli are N-Ž2X-deoxyguanosin-8-yl.-1-amino-8-nitropyrene w39x. Thus, it seems plausible that, unlike pyrimidine dimers or abasic sites, the DNA adduct induced by 1,8-DNP does not severely block DNA replication when it is formed at the CG repetitive sequence. 1,8-DNP exhibited low, but significant, mutagenicity in the strain YG5147 harboring vector plasmids alone, suggesting that DNA polymerases can bypass the mutagenic DNA lesion at the CG repetitive sequence, to some extent, even in the absence of mutagenesis proteins. The results obtained with 2-NF and Glu-P-1 Žgroup 3. suggest that these compounds induce mutations without the assistance of the mutagenesis proteins ŽTable 2 and Fig. 1.. This conclusion is in accordance with the report that the mutagenicities of 2-NF and Glu-P-1 in S. typhimurium D3052 are not affected by the deletion of umuDCST genes w24x. It is likely that the DNA adducts generated by 2-NF and Glu-P-1 do not strongly block replication and the DNA polymerase itself can easily bypass these structures by a slippage mechanism. It is indeed reported that the DNA polymerase III holoenzyme replicates
39
DNA template containing N-Ždeoxyguanosin-C8-yl.2-aminofluorene, which is thought to be formed from the metabolically activated 2-NF, with high efficiency w40x. This DNA adduct is also formed by 2-aminofluorene Ž2-AF ., following oxidative metabolism. Recently, it was reported that the products of umuDC are not required for frameshift mutagenesis by IQ. IQ–DNA adducts behave more similarly to 2-NF or 2-AF adducts than acetylaminofluorene ŽAAF. adducts, which strongly hinder DNA replication w28x. Since the expression of the mutagenesis proteins in our plasmids is controllable by IPTG w32x, we examined the effects of their levels of expression on the mutagenesis. The expression level should be similar between SamAX and MucAX and between SamB and MucB because the expression from all the AX and B genes is controlled by the identical Shine– Dalgarno sequence linked to the identical lac promoter. Interestingly, IPTG treatments exhibited opposite effects on the mutabilities of the strains expressing SamAX B or MucAX B. In strains harboring the plasmids expressing SamAX B, IPTG treatments increased the mutabilities of the host strains to furylfuramide, AFB1, 1-NP and 1,8-DNP ŽTable 3.. This is consistent with the previous knowledge that samAB on multi-copy-number plasmids more efficiently promotes UV mutagenesis than that on low-copy-number plasmids w9,31x. UmuC-like proteins are very unstable and tend to aggregate w14,41x. SamB may be less stable than MucB, and thus a higher level of expression of SamAX B is necessary to carry out the extent of mutagenesis promoted by a lower level of expression of MucAX B. In contrast, in the strains harboring the plasmids expressing MucAX B, IPTG treatments reduced the mutabilities of the host strains for all the chemicals tested ŽTable 3.. In particular, IPTG treatment substantially reduced the mutability by furylfuramide of the strains expressing MucAX B to furylfuramide ŽTables 2 and 3.. Interestingly, it is reported that expression of excess UmuC results in a non-mutable phenotype of E. coli w42x. Mutagenesis proteins, such as SamAX B, MucAX B or UmuDX C, could have optimum intracellular concentrations for mutagenesis, and they might rather decrease the mutagenesis if they are expressed too much. It could be possible, however, that this apparent reduction of mutagenesis by the elevated expression of MucAX B
40
P. Gruz et al.r Mutation Research 398 (1998) 33–42
is due to the killing of host cells rather than the real reduction of mutagenesis because it has been known that the overexpression of UmuDC is associated with cell lethality w43x. It is also reported that overexpression of UmuDX C results in the in vitro formation of UmuDX C–ssDNA complex, which blocks DNA replication w41x. A surprising observation was that the elevated expression of MucB alone could promote the frameshift mutagenesis induced by 1-NP and 1,8DNP ŽFig. 2.. It is conceivable that DNA polymerases associated with MucB, without the assistance of MucAX or SamAX , can bypass the lesions induced by nitropyrenes. It is proposed that RecA targets the MucAX or UmuDX to DNA lesions, thereby allowing the complexes of MucAX B or UmuDX C to interact with DNA polymerases at the lesions w16x. MucAX and UmuDX proteins interact with RecA as well as MucB and UmuC, respectively w44x. Thus, MucB protein will not be well targeted to DNA lesions if MucAX is absent. The results shown in Fig. 2 raise the possibility that a trace amount of MucB protein alone is sufficient for supporting translesion bypass of DNA polymerases at the lesions induced by nitropyrenes. It could also be interesting to examine whether the MucB protein requires the presence of RecA for such AX-independent enhancement of nitropyrene-induced mutagenesis. There is also another conceivable explanation for this AX-independent enhancement. The chromosome or 60-MDa cryptic plasmid of S. typhimurium might encode some yet undiscovered UmuD-like protein that is able to target the MucB protein to the nitropyrene-modified DNA. For instance, UmuDX-like protein termed HumD was found in the genome of bacteriophage P1 w45x and an UmuC-like protein termed DinPrB has been recently cloned from the genome of E. coli w46x. Such Umu-like proteins might interact with mutagenesis proteins and modulate their activity under certain circumstances. The expression of SamAX B or MucAX B did not significantly increase the number of spontaneous Hisq revertants of the hisD3052 allele ŽTable 4.. This is in contrast with the previous results that the expression of MucAX B markedly increases the number of spontaneous Hisq base-substitution revertants of the hisG428 or hisG46 alleles w32x. Spontaneous frameshift mutations mainly occur by slippage of
DNA replication at repetitive DNA sequences w47x. S. typhimurium hisD3052 allele has a CGCGCGCG sequence, which is a hot spot for spontaneous as well as chemically induced frameshift mutations w24x. Thus, we suggested that neither SamAX B nor MucAX B efficiently promotes the slippage error of DNA replication at the repetitive sequences. Alternatively, the spontaneous mutations might occur at repetitive sequences having endogenous DNA lesions that do not strongly hinder DNA replication as in the case of chemicals of group 3 in this study w48x. In summary, we have demonstrated that the elevated expression of SamAX B can promote frameshift mutagenesis by furylfuramide, AFB1, 1-NP and 1,8DNP with efficiencies comparable to or even higher than that of MucAX B. The abilities of SamAX B and MucAX B to promote mutagenesis strongly depended on the levels of their expression. We propose that B-proteins rather than AX-proteins play a crucial role in mutagenesis because the elevated expression of MucB alone could promote frameshift mutagenesis by 1-NP and 1,8-DNP.
Acknowledgements We thank Dr. P.D. Josephy, University of Guelph, for critically reading the manuscript. This work was supported by grants-in-aid for Scientific Research on Priority Areas Ž08280104. from the Ministry of Education, Science, Sports and Culture of Japan, Science and Technology Agency of Japan, and the Japanese Health Science Foundation. P.G. was supported by fellowships from the Japanese Health Science Foundation and the Science and Technology Agency.
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