Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions

Mutation Research, 268 (1992) 59-64 © 1992 Elsevier Science Publishers B.V. All rights reserved 002 5107/92/$05.00

59

MUT 05100

Analysis of phage M13mp2 mutants produced from transfection of phage D N A having N4-aminocytosines at defined sequence positions Keiko Matsumoto, Tetsuya Yashiki, Tadayoshi Bessho, Kazuo Negishi a and Hikoya Hayatsu Faculty of Pharmaceutical Sciences and a Gene Research Center, Oka~ama Unicersity, Tsushima, Okayama 700 (Japan) (Received 4 August 1991) (Revision received 17 December 1991) (Accepted 19 December 1991)

Keywords: Nucieoside analog; N4-Aminodeoxycytidine5'-triphosphate; DNA polymerase I; Site-directed mutagenesis

Summary N4-Aminocytidine is mutagenic in various organisms. In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA. To prove that the NLaminocv'.osine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZa region, by carrying out in vitro limited extension of primed phage DNA. We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations. The M! 3rap2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies. Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present.

N4-Aminocytidine is a potent mutagen of a nucleoside (base) analog type. We have reported that N4-aminocytidine is mutagenic to bacteriophages, prokaryotes, and eukaryotes (Negishi et al., 1983, 1985; Nomura et al., 1987; Takahashi et al., 1988). The analysis of the mutation spectrum in M13mp2 phage showed that the analog can induce both AT-to-GC and GC-to-AT transitions (Bessho et al., 1989). No transversions, deletions

Correspondence: Dr. H. Hayatsu, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700 (Japan). Tel.: (0862) 52-1111 ext. 995; Fax: (0862) 54-2129.

or insertions were observed. When N4-aminocytidine was administered to Escherichia coli or mammalian cells growing in culture, the DNA isolated from the cells after growth was found to contain N4.aminodeoxycytidine residues (Nomura et al., 1987; Negishi et al., 1988). This finding indicates that N4-aminocytidine should be metabolized in the cell into N4-aminodeoxycytidine 5'-triphosphate (dC'~mTp) (Fig. 1), which will then be incorporated into DNA. We have also reported that E. coli DNA polymerase I and mammalian DNA polymerase a can catalyze the incorporation of dc'~mTp into DNA in vitro, efficiently in place of dCTP opposite

H2NNH

O" I

O" I

O" I

"O-P-O--P-O-P-O----,

i

0

I

OH Fig. 1. N4-Aminodeo~3,cytidine 5'-triphosphate.

guanine and less efficiently, but to a significant extent, in place of dTTP opposite adenine (Negishi et al., 1985; Takahashi et al., 1988). N4-Aminodeoxycytidine thus incorporated in DNA template directs the incorporation of dGTP. These experiments indicate that the N4-amino cytosine-containing DNA can cause AT-to-GC transitions, indeed it was shown that incorporation of dCamTP into the replicative form DNA of phage ~X174am3 by nick translation can lead to the production of revertant phages (Takahashi et al., 1985). The presence of GC-to-AT transitions in the mutation spectrum (Bessho et al., 1989) dictates that N4-aminocytosine in template should direct incorporation of dATP. However, our previous biochemical analysis has not provided evidence for this incorporation, in the present work, we have attempted to prove this mutational pathway and to show that the site of N4-aminocytosinc residues in the original DNA is the site of mutagenesis in the DNA of the progeny. Thus we carried out Klenow enzyme-catalyzed incorporation of dC~'mTP into defined sites of DNA, using single-stranded DNA of phage M13mp2 as template and allowing limited chain elongation of a series of priming oligodeoxyribonucleotides that were annealed to the template at the lacZa region. Mutations took place at positions where N4-aminocytosine residues were originally incorporated into the minus strand of the replicative form DNA and that these mutations were transitions of either of the Ai'-to-GC and GC-to-AT types. Materials and methods

Materials dC"mTp was prepared as previously described (Negishi et al., 1985). E. coli DNA polymerase I

large fragment was obtained from Takara (Kyoto, Japan). E. coli NR9099 and phage M13mp2 were gifts from Dr. T.A. Kunkel (Kunkel, 1985). A mutant phage used for the reversion assay was a strain prepared in our previous experiments (Bessho et al., 1989). Oligonucleotides used for primers were gifts from Drs. T. Takeda and M. Yamamoto of the Institute of Medical Sciences, University of Tokyo, who synthesized them in an Applied Biosystems Model 380B DNA synthesizer.

Mutagenesis M13mp2 single-stranded DNA, 0.4 pmole, used as a template was annealed to 1 pmole of a primer with heating at 60°C for 15 min, followed by cooling to room temperature, in 10 /~l of a solution containing 10.5 mM Tris-HCl (pH 7.5), 30 mM NaCl, 10.5 mM MgCl 2 and 1.5 mM EDTA. For the incorporation of dC"mTp, 10 p,l of the reaction mixture composed of 0.08 pmole of the template primer, 16/~M each of dC"mTp and of two kinds of normal dNTPs, and 2 units of DNA polymerase I large fragment from E. coli (Klenow enzyme) was incubated at 37°C for 20 min. Then 1.1 /zl of a solution containing l mM of each of the four normal dNTPs was added and the mixture was incubated at 37°C for an additional period of 20 rain. A portion of the products was transfected into the Ca2+-treated E. coli NR9099, which carries a chromosomal deletion in the iac operon and contains F' plasmid having a defective /3-galactosidase gene. The transfected bacteria were plated with growing E. coli. Colorless and light blue plaques, which the mutant progeny phages produced, and the blue plaques, that were of the wild type, were scored to measure the mutation frequencies. The DNA of the mutants was isolated and the sequence was determined with the dideoxy procedure (Sanger et al., 1977) as described previously (Bessho et al., 1989). Analysis of elongated products The oligonucleotides used as the primers were labelled at the 5' end with y-'~2p-ATP and T4 polynucleotide kinase using the Megalabel kit obtained from Takara. The incorporation of dC~mTp was done under the conditions described above for the mutagenesis except that 0.2

61 pmole o f the template p r i m e r was used. T h e p r o d u c t s were d e n a t u r e d with heat and analyzed with 2 0 % polyacrylamide gel electrophoresis und e r d e n a t u r i n g conditions.

TABLE I PRIMERS USED IN THE KLENOW ENZYME-CATALYZED POLYNUCLEOTIDE SYNTHESIS Primer No.

Sequence

Results

5' 6GCCTCTTCGCTATTACG

3'

Incorporation of dCamTp

5' 5' 5' 5~

3' 3' 3' 3'

Fig. 2 shows the sequence of the portion of M13mp2 single-stranded DNA lacZa region on which complementary oligonucleotides (Nos. 1-5 in Table 1) were annealed and used as the primers of Klenow enzyme-mediated polynucleotide synthesis. First, the polynucleotides obtained on elongating 32p-labeled primer 1 were analyzed. When dCTP was the only triphosphate substrate used in the synthesis, the 18-nucleotide primer was elongated to a 20-mer (Fig. 3, lane 2). When dC"mTp was used in place of dCTP, a similar elongation took place (lane 3). When dC"mTp (or dCTP) and dATP were the substrates, a 21-mer was the major product (lanes 5 and 4). With primer 2, again an elongation of the 18-mer to a 20-mer occurred when dCTP or dC"mTp was used as the sole substrate (lanes 10 and 11). Addition of dATP and dGTP to the incubation mixture resulted in a further elongation to give a 31- (or 32-)mer as the product (lane 12). For unknown reasons, the oligomers bearing C "m migrated slightly behind the unmodified oligomers. These results show that N4-aminocytosine nucleotide can be incorporated opposite guanine of the template strand, and that with this procedure it may be possible to produce a double-stranded form of the phage DNA having N4-aminocytosine residue(s) at specified position(s) of the daughter strand.

5'-GATAACAA'IT TCACACAGGA AACA(;CTATG ACC AI"(: ..........

~-~0"

r~,,

Aa'r 'rCA CT(; (;CC (;T¢: (;'rT TTA CA.A.t'(.;'f. L'(.;.T..(.;.A(_;.'[(.;G. (;AA .90

AAC CCT G G C ( ; T T ACC I.'AA CTT AAT C G C CTT GCA GCA CAT '~o 2 -120

CCC E E l ' TTC GCC AGE '.r.(.;(.;. CGT AAT AGC (:AA GAG (;('C C(;C-3'

.150

Transfection of DNA bearing N~-aminocytosines at specified positions dc'~mTp was i n c o r p o r a t e d during the elongation of primers 1 - 5 with the catalysis o f Klenow enzyme on the t e m p l a t e of M13mp2 D N A . In this process, only two out o f the three normal nu-

Primer-1

Primer-2

1 2 3 4 5 6 7 8 9101112 13 C C*C C* C C* C C'C* A A A A A T T O

==Dalb

O

~ll= O

AT'[ ACG Nt~ 4

*60

GGTAACGCCAGGGTTTTC ATCATGGTCATAGCTGTT ACGCCAGGGTTTTCCCAG ¢TCTTCGCTATTACGCCA

N,~ I

Fig. 2. Primer attachment sites in M13mp2 DNA. Dotted lines indicate positions where the limited elongation of primers will take place.

Fig. 3. Elongation of ~2P-labeled primers annealed to singlestranded MI3mp2 DNA using dc"mTp or dCTP as a substrate with and without other substrates. Primer No. 1 (lanes | and 6) was elongated in lanes 2, 3, 4, 5, 7 and 8. Primer No. 2 (lanes 9 und 13) was elongated in lanes 10, 11 and 12. Substrates used in the elongation: lanes 2 and 10, dCTP; ianes 3 and 11, dCamTp; lane 4, dCTP and dATP; lane 5, dCamTp and dATP; lane 7, dCTP, dATP and dTI'P; lane 8, dc"mTp, dATP and dTTP" lane 12, dCumTp, dATP and dGTP. The migration of oligonucleotides was from the top to the bottom of the figure.

62 A

cleotides, i.e., dATP, dGTP and dTTP, were supplemented to dcamTp. For controls, we used dCTP in place of dCamTp. The sites of N 4aminocytosine incorporated were in this way limited at specific positions. Fig. 2 shows the nucleotide positions where this limited elongation of DNA synthesis was expected to occur. These limited elongations were followed by an exhaustive elongation (chasing) with four normal dNTPs added in large excesses. The DNA thus prepared was transfected to E. coil and mutations in progeny phages were analyzed. As shown in Table 2, significantly higher mutation frequencies were found for DNAs bearing N4-aminocytosines as compared with those for the control DNAs. Thus, the induced mutation frequencies increased from < 2 × 10 - 4 in the control samples to 2-11 x 10 -3 in the N 4aminocytosine-containing samples. We randomly selected 31 mutants from those obtained in the experiments using primers 1 and 2, and their DNA sequences were analyzed. Among them, 25 mutants were found to have sequence alterations in the regions proximate to the 3' terminal of the annealed priming oligonu-

TABLE 2 MUTANT PHAGES GENERATED BY TRANSFECTING MI3mp2 DNA TO E, coil Procedure for preparing Ml3mp2 DNA used in transfection

Number of mutants/10 4 plaques formed

Primer No.

dNTP used in the limited elongation of primer a

1 1

C am, A, T C, A , T

39 0

2 2

C am. A, G C,A,G

110 2

3 3

C ~'m,G. T C,G.T

23 2

4 4

C ~". A, T C, A.T

33 0

5 5

C ~'m.G, T C,G,T

37 0

a The limited elongation was followed by chasing with added dCTP, dTFP, dATP, and dGTP. C am represents N 4aminocytosine

5' GCC AGC T G G C G T A A T A G C G A A G A G G C C 3' No. I A 4

Mutant sequenO:: No, of m u t t , s :

5' CAA C G T C G T ~. Mutant sequence: G No, ofmutams: 3

GAC T G G G A A A A C C C T G G C G T T A C C 11~ ~.1~ No. 2 AG AA I 18 1 3

3'

B. D N A seque,cesof mum.ms, derivedfrom primer-2,withdaublebase changes Mulam I Mutant2 Mutant3 Muta.nz4 Mutam5

5'C A A 5' C A G 5' C A G 5' CA(3 5'CAA

CGT CGT CGT CGT CGT

CGT CGT CGT CGT CGT

GAC GGC GGC G.~.C AGe

TAA TGG TGG TGG TGG

GAAAACCCTGGcG'rTACC GAAAACCC'I['GGCGII'rACC GAAAACCCTGGCGTTACC GAAAACCCTGGCGTTACC GAAAACCCTGGCGTTACC3'

3' 3' 3' 3'

Fig. 4. Sequence alterations in DNA of the mutants arising from incorporation of dCamTp into primers No. 1 and No. 2 annealed to M13mp2 single stranded DNA. See Table 2 for elongation conditions. (A) Distribution of base changes. Arrows in the figure indicate the positions of base alterations, corresponding bases in sequences of the mutant DNA and yields of the mutations. (B) The sequences of the induced mutants bearing double base changes. The altered bases are underlined.

cleotides: 20 mutants with single base changes and five mutants with double base changes. Fig. 4 shows these sequence changes. There were 30 base alterations in total. As expected, all the mutations were transitions, A-to-G and G-to-A in viral strands. In the experiment using primer 2, the mutation frequency observed was very high, with mutation sites detected as far apart as 13 bases from the 3' end of the primer. This phenomenon, in contrast to the results obtained with primer 1, is clearly derived from the use of a combination of dCamTp, dATP and dGTP as the substrate set. In the experiments with the other primers, only a single kind of purine nucleotide was present as the supplementary nucleotide to dCamTp (Table 2). Thus, the dual character of dC~mTp as a substitute for both dCTP and dTI'P (Negishi et al., 1985) allowed the elongation of primer 2 to proceed up to 13 bases (Fig. 3, lane 12), the production of a mutant DNA thereby becoming more frequent. With the use of the other primers, the incorporation of N4-aminocytosine would have been restricted within smaller regions flanking the 3' end of the primers (Fig. 2), and therefore, the lower mutation frequencies observed for these samples were reasonable. Sequence analysis of

63 Mutant sequence: 5" GCC AGe TGA CGTAATAGCGAAGAGGCC 3' No. I Revertant sequence: G Fig. 5. Reversion o f a m u t a n t phage.

mutants obtained with the use of primer 1 showed that the mutations occurred only at the nucleotide next to the 3' end of the primer. Reversion induced with the use of dCa'TP The induction of mutation at the N4-aminocytosine site in DNA was further demonstrated with a reversion assay. The sequence of the mutant DNA used is shown in Fig. 5. Single-stranded DNA from a mutant M13mp2 phage bearing a G-to-A transi:ion was annealed to primer 1, with the annealing position starting one nucleotide downstream from the transition site. The template primer was incubated with Klenow enzyme using dCamTP as the only nucleoside triphosphate added, and then chased. Transfection assay of the DNA revealed that the mutation frequency increased to a high value, 16 x 10 -3 of total progeny phages (Table 3). The DNA sequence analysis of three revertants obtained showed that their sequences were the same as that of the wild type, i.e., an A-to-G reversion took place at the position of the original mutation. This induction of mutation was abolished by addition of d'ITP during the initial elongation step. dTTP can be expected to compete with dC"mTp in the incorporation at the site opposite the adenine, in contrast, the addition of dCTP, a non-competitive nucleotide, had very little effect (Table 3).

TABLE 3 REVERSION OBTAINED BY TRANSFECTING DNA PREPARED FROM SINGLE-STRANDED MUTANT Ml3mp2 DNA dNTP used in the limited elongationof primer

Numberof revertants/ 104 plaques formed

C am C am, C C :'m, T C am, ", T C C, T, G, A

160 110 0 0 0 0

Discussion

The present results indicated that N4-amino cytosines incorporated in place of cytosine direct the incorporation of dATP. The other possible mechanisms for the induction of G-to-A transitions by the N4-aminocytosine incorporated in place of cytosine is that spontaneous deamination of N4-aminocytosine to form uracil would lead to incorporation of dATP opposite the position in the next replication at the site. To evaluate the latter possibility, dCamTp was singly used as the substrate for elongating primer 2, thereby allowing the formation of a 20-mer (Fig. 3, lane 11). Then, after the chase reaction was carried out, the solution, which contained the polynucleotide formed, was incubated additionally at 37°C for the possible deamination and used for the transfection assay. The additional incubation, however, caused no further increase in the mutation frequency: the mutation frequencies found were 5.7 × l0 -3 at time 0, 4.2 × 10 -3 at 1 h, 6.6 × 10 -3 at 3 h, and 4.6 × 10 -a at 5 h. Therefore, it is unlikely that the mutation was induced by spontaneous deamination of N4-aminocytosine residues in the DNA. The yields of mutants from the experiment using primer 2 (Fig. 4) show that A-to-G predominates over G-to-A: a total of 21 A-to-G changes in two sites (average, 10.5 per site) and 5 G-to-A changes in three sites (average, 1.7 per site). This in turn suggests that the efficiency of dGTP incorporation at a site opposite N4-aminocytosine in the template should be much higher than that of dATP incorporation, provided that there are no additional discriminating processes in the bacterial cells. This observation is consistent with our previous finding that the ratio between the incorporation efficiency of dC"mTp into a site opposite guanine in template and that opposite adenine in template is 12/1 (Negishi et al., 1985). It also agrees with the tautomeric constant, 30/1, estimated by Brown et al. (1968) for the equilibrium between the amino and the imino forms of l-methyl-N 4. lminocytosine and consistent with our hypothesis that the abundant amino form of Na-aminocytosine pairs with guanine and the rare imino form pairs with adenine (Negishi et al., 1985). It is possible that in this ambiguous base-

64

pairing, other mechanisms such as ionized and wobble-type pairing are also involved, as in the case of 2-aminopurine and 5-bromouracil (Sowers et al., 1986, 1989). Mutagens of base-analog types have been known since the early phase of research on mutagenesis. The mutagenesis is believed to be caused by replicational errors, as proposed by Freese (1959). Generally, base analogs have been shown to be incorporated into DNA. However, only demonstrating the incorporation is not sufficient to prove this mechanism; the mutagenic potential of the DNA containing the analog should be demonstrated. Here we have shown that the DNA having N4-aminodeoxycytidine 5'-triphosphate incorporated in it does cause mutation. Furthermore, the results have clearly shown that base changes in the mutant DNA take place at positions where the base analog was originally present. This type of mutagenesis can have a practical potential. N4-Hydroxydeoxycytidine 5'-triphosphate, a putative metabolite of N4-hydroxycytidine, was shown to be very mutagenic and has been used extensively in in vitro mutagenesis experiments (Wieringa et al., 1983). Incorporation of 5-bromodeoxyuridine 5'-triphosphate into phage M13mpl0 at a specified gene sequence resulted in the induction of mutations in that gene (Muller et al., 1988). The present results indicate that N4-aminodeoxycytidine 5'-triphosphate can be used for inducing AT-to-GC and GC-to-AT mutations in a desired region of a gene.

Acknowledgments This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan (02151039 to K.N. and H.H.). The radioisotope experiments in the present work were carried out in the Radio-Isotope Laboratory of Okayama University. References Bessho. T., K. Matsumoto, A. Nomura, H. Hayatsu and K. Negishi (1989) Spectrum of N4-aminocytidine mutagenesis, J. Mol. Biol., 205, 659-664.

Brown, D.M., M.J. Hewlins and P. Schell (1968) The tautomeric state of N(4)-hydroxy- and N(4)-amino-cytosine derivatives, J. Chem. Soe. (C), 1925-1929. Freese, E. (1959) The specific mutagenic effect of base analogues on phage T4, J. Mol. Biol., 1, 87-105. Kunkel, T.A. (1985) The mutational specificity of DNA polymerase-/3 during in vitro DNA synthesis, J. Biol. Chem., 260, 5787-5796. Muller, M., J. Martial and W.G. Verly (1988) Pairing properties of bromouracU and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis, Biochem. J., 253, 637-643. Negishi, K., C. Harada, Y. Ohara, K. Oohara, N. Nitta and H. Hayatsu (1983) N4-Aminocytidine, a nucleoside analog that has an exceptionally high mutagenie activity, Nucleic Acids Res., 11, 5223-5233. Negishi, K., M. Takahashi, Y. Yamashita, M. Nishizawa and H. Hayatsu (1985) Mutagenesis by N4-aminocytidine: induction of AT to GC transition and its molecular mechanism, Biochemistw, 24, 7273-7278. Negishi, K., K. Tamanoi, M. Ishii, M. Kawakami, Y. Yamashita and H. Hayatsu (1988) Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coil, J. Bacteriol,, 170, 5257-5262. Nomura, A., K, Negishi, H. Hayatsu and Y. Kuroda (1987) Mutagenicity of N~-aminocytidine and its derivatives in Chinese hamster lung V79 cells: incorporation of N 4. aminocytosine into cellular DNA, Mutation Res., 177, 283-287. Sanger, F,, S. Nikelen and A.R. Coulson (1977) DNA sequencing with chain-terminating inhibitors, Proc, Natl. Acad, Sci. (U.S.A,), 74, 5463-5467. Sowers, L.C., G.V. Fazakerley, R. Eritja, B.E. Kaplan and M.F. Goodman (1986) Base pairing and mutagenesis: ob. servation of a protonated base pair between 2-aminopurinc and cytosine in an oligonucleotide by proton NMR, Proc. Natl. Acad. Sci. (U.S.A.), 83, 5434-5438. Sowers, L.C., M.F. Goodman, R. Eritja, B. Kaplan and G.V. Fazakerley (1989) Ionized and wobble base-pairing for bromouracil-guaninc in equilibrium under physiological conditions, J. Mol. Biol,, 205, 473-477. Takahashi, M., K. Negishi and H. Hayatsu (1985) Induction of mutation in vitro in phage ~bX174 am3 by N 4aminodeo~cytidine triphosphate, Biochem. Biophys. Res. Commun., 131, 104-109. Takahashi, M., M. Nishizawa, K. Negishi, F. Hanaoka, M. Yamada and H. Hayatsu (1988) Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors, Mol. Cell, Biol., 8, 347-352. Wieringa, B., F. Meyer, L Reiser and C. Weissmann (1983) Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit/]-globin gene, Nature (London), 301, 38-43.