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Copyright 0 1985 by the Genetics Society of America

D N A SEQ U EN C ES OF F R A M E SH IF T A N D O T H E RM U T A T I O N S I N D U C E D BY ICR-170 IN YEAST

JOACHIM F. ERNST,**' D . MICHAEL HAMPSEY* AND FRED SHERMAN*-t***Department of Radiation Biology and Biophysics and +Department o Biochemistry, University o

Rochester School of Medicine an d Dentistry, Rochester, New York 14 64 2Manuscript received April 27, 1985

Accepted June 14, 1985

ABSTRACTICR-170-induced mutations in the CYCl gene of the yeast Saccharomyces

cerevisiae were investigated by genetic and DNA sequence analyses. Geneticanalysis of 33 cycl mutations induced by ICR-170 and sequence analysis ofeight representatives demonstrated that over one-third were frameshiftmutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly,and a few of these were not frameshift mutations. The sequence results indicatethat ICR-170 primarily induces G.C additions at sites containing monotonousruns of three G.C base pairs. However, some ' . sites within the CYClgene were not mutated by ICR-170. Thus, ICR-170 is a relatively specificmutagen that preferentially acts on certain sites with monotonous runs of G .C base pairs.

-G-G-G--c-c-c-

HE iso-1-cytochrome c system of the yeast Saccharomyces cerevisiae has beenT used to investigate the alterations induced by a variety of physical andchemical agents. Procedures for selecting series of mutations of the type C Y C l ++ ycl-x +- YCl-x-y have been described, where C Y C l + denotes the wild-typeallele encoding iso- 1 cytochrome c, cycl -x denotes mutations causingdeficiencies or loss of function of iso-1-cytochrome c and CYCI-x-y denotesintragenic reversions producing functional iso- 1 cytochromes c that are eithernormal or altered (SHERMANt al. 1974, 1975). By determining the aminoacid sequences of iso-1-cytochromes c from intragenic revertants, thenucleotide changes of many cycl mutations, as well as many C Y C l reversions,have been deduced (for reviews, see SHERMANnd STEWART 971, 1973,1978). More recently, current techniques of cloning and D N A sequencing wereused to directly determine the nucleotide changes of cycl mutations and their

Abbreviations used in text: ICR-170, 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)minopropyl-aminolacridine HCI; ICR-I 91, 2-chloro-6-methoxy-9-[3-(2-chloroethyl)minopropylamino]acridineHCI.' Present address: Biogen S. A., Route des Acacias 46, 1227 Carouge, Geneva, Switzerland.To whom correspondence should be addressed.

Genetics 111: 233-241 October, 1985.

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234 J. F . E R N S T , D. M . H AM P SEY AND F . SH ERM ANrevertants (ERNST, STEWARTnd SHERMAN981, 1982; ERNST et al. 1985;STILESt al. 1981; ZARET and SHERMAN982, 1984).

In this investigation, we have used these DNA procedures to determine thenucleotide changes of c y c l mutations induced with the mutagen ICR-170.Unlike c y c l mutations induced with most other mutagens, over one-third ofthe cycl mutations induced with ICR-170 mapped at a single site in the C Y C lgene (SHERMANt al. 1975). The DNA sequences demonstrate that ICR-170induces primarily additions of G C base pairs at sites containing monotonousstretches of three G - C base pairs and that one such sequence is preferentiallymutated.

M A T E R IA L S A N D M E T H O DSRetrieving and DNA sequencing of most of the cycl mutations were accomplished by the

procedures described by ERNSTet al. (1985). These procedures require the use of the plasmidspAB61 or pAB178, which can replicate and be selected for in both yeast and Escherichia coli andwhich can also integrate adjacent to the CYCl locus. Rather than introducing pAB61 or pAB178by transformation into each strain containing a cycl-x mutation whose sequence is desired, eitherof these plasmids i s first transformed into a haploid yeast strain lacking the CYCl chromosomalregion. These strains are then used to introduce the plasmid into each haploid cycl-x strain by asimple cross. After the plasmid integrates adjacent to the cycl-x mutation in the diploid strain, thegenomic DNA is digested with BamHI, a restriction endonuclease that will excise a major portionof the integrated plasmid along with the chromosomal segment containing the cycl-x mutation.The fragments are ligated, and the plasmid with the cycl-x segment is recovered by transformationof E. coli. The cyc l-x mutation can be conveniently sequenced by the standard dideoxy-terminatormethod, using one or more of five small synthetic primers homologous to various sites distributedon both strands of the CYCl gene (ERNSTet al. 1985).

RESULTSPreviously, SHERMANt al. (1974, 1975) described the isolation and genetic

characterization of 195 cycl mutations, which included 28 mutations inducedby ICR-170. These studies have been extended and a total of 464 c y c l muta-tions now have been isolated and characterized, including 33 induced by ICR-170 (F. SHERMANnd S . C O N S A U L , unpublished results). The results, summa-rized in Figure 1, indicate that 12 of the ICR-170-induced mutants map atthe c y c l - 1 5 site, which was previously shown to correspond to a site at oraround amino acid position 29; the remaining 21 are more-or-less randomlydistributed along the C Y C l locus. Cloning and DNA sequencing were under-taken with the four mutations c y c l - 1 5 , c y c l - 1 6 , c y c l - 1 9 1 and c y c l - 2 0 1 , whichmap at the same site that was at or near amino acid position 29, and with thesingle mutations c y c l - 4 2 , c y c l - 1 8 9 , c y c l - 1 9 0 and c y c l - 2 1 1 , which map, respec-tively, at sites at o r around amino acid positions 85, 64 , 38 and 76. The DNAsequences of these eight c y c l mutations are presented in Table 1. An exampleof a sequencing gel is shown in Figure 2, where the c y c l - 1 5 alteration iscompared to the normal C Y C l f sequence. The DNA sequences establishedthat the four mutations c y c l - 1 5 , c y c l - 1 6 , c y c l - I 9 1 and c y c l - 2 0 1 are identicaland contain an insertion of a G.C base pair at the site corresponding to aminoacid positions 29-30, as shown in Figure 3. Similarly, the DNA sequence

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ICR- 170-INDUCED MUTATIONS IN YEAST 235

Amino acid positionFIGURE .-The assignments of 464 cycl mutations to each of the mutational sites [from SHER-

M A N et al. (1975) and F. SHERMANnd S. CONSAUL, npublished results]. Nonsuppressible mutantscompletely, or almost completely, lacking iso-1-cytochrome c are indicated by 0; those havingdetectable amounts of iso-I-cytochrome c are indicated by 0 , and mutations suppressible by UAAan d U A G suppressors are designated by A. Mutations induced by ICR-170 are denoted by thesame symbols but are filled in. T h e other mutations arose spontaneously or were induced by UV(ultraviolet light), N I L (l-nitrosoimidazolidone-2),QO (4-nitroquinoline-l-oxide),EB (diepox-ybutane), NA (nitrous acid) or EM S (ethyl methanesulfonate).established that the c y c l - 2 1 1 mutation contains an extra G - C base pair at asite corresponding to amino acid positions 75-76 (Figure 3). Thus , all f ive ofthese ICR-170-induced mutations arose by insertion of a G C base pair, andthe insertions occur at sites normally containing a -c.-&c..-sequence. Bycontrast, both the c y c l - 4 2 and c y c l - 1 9 0 mutations arose by A . T -+ C - G trans-versions, and the c y c l - 1 8 9 mutations arose by a G - C+ T - A transversion.

-G-G-G-

DISCUSSIONThe spectrum of DNA alterations induced by ICR-170 was determined by

genetic analysis of cycl mutations and by DNA sequencing of eight represent-ative mutations. DNA sequences established that the four mutations cycl -15,cyc l -16 , cyc l -191 and c y c l - 2 0 1 are located at the site corresponding to aminoacid positions 29-30, that the single mutation c y c l - 2 1 1 is located at the sitecorresponding to amino acid positions 75-76 and that all of these cycl muta-tions arose by insertion of a G - C base pair at a monotonous stretch of threeG.C base pairs, as shown in Figure 3.

As shown in Figure 1, 12 out of 33 ICR-170-induced mutations are at thec y c l - 1 5 site. No more than two ICR-170 mutations map at any of the othersites. DNA sequence and genetic analyses indicate that not all ICR-170-inducedmutations ar e frameshift mutations and that some do not map at sites contain-ing stretches of G C base pairs. The ICR-170-induced mutations include threemissense mutations, c y c l - 4 2 , c y c l - 1 8 9 and c y c l - 1 9 0 , at positions 85, 64 and 38,respectively, and one UAA mutation, c y c l - 2 0 2 , at position 21. DNA sequencingestablished that the c y c l - 4 2 and c y c l - 1 9 0 missense mutations arose by A.T -+C - G tranversions, resulting in Met85 + Arg85 and His38 + Pro38 substitu-tions, respectively, whereas the cycl -189 missense mutation arose by a G - C --.*T - A transversion, resulting in a T rp64 + Cys64 substitution (Table 1; also

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236 J. F . E R N S T , D . M . H A M P S E Y A N D F . S H E R M A NTABLE 1

DNA sequences on cyc l mutations induced with ICR-170Allele Sequen ce change" Mutational change Sequencing procedureb

c y c l - 15

cycl-16

cyc l I 9 I

c y c l - 2 0 1

c y c l - 2 1 1

c y c l - 4 2

cycl-189

cycl- I90

90ccc90ccc90ccc90ccc228ccc257ATG

195TGG116CAT

I

I

I

I

I

I

I

I

90+ cccc

90+ cccc

90+ cccc

90+ cccc

I

I

I

I228I

+ cccc257I

+ AGG195

+ TGTI116

I+ CCT

+ G.C+ G.C+ G.C+ G.C+ G.CA . T + C.G

G . C -+ T * A

A.T + C.G

Primers A an d D

Primers A and D

Primer D

Primers A and D

Primers C an d E

Primer E

Primer C

Primer Da T h e positions of the nucleotides of the CYCl gene are shown in Figure 3.T h e DNA sequences were determined by the procedure of ERNST t al . (1985), using theindicated primers.

see SCH WEING RUBER, STEWARTnd SHERMAN979). Fine-structure mappingand tests with suppressors suggest that the UAA mutation c y c l - 2 0 2 was formedfrom the CAA codon of Gln21 by a G- C+ A.T transition.

The examination of the D N A sequence of the normal CYCI' gene indicatesthat not all contiguous G C stretches are particularly mutable with ICR-170.As shown in Figure 3, there are six sites containing monotonous stretches ofthree G -C base pairs. The cyc l - 1 5 and 11 other mutations mapped at the siteat positions 29-30; the cyc l - 2 1 1 mutation mapped at positions 75-76; the cyc l -196 and c y c l - 2 0 0 mutations mapped in the region of positions 27-28; and thec y c l - 7 0 5 mutation mapped in the region of position 89. Also, it should benoted that the ICR-170-induced mutation, c y c l - 1 8 9 , was a base-pair substitu-tion within a stretch of three G - C base pairs at positions 64-65. However,none of the ICR-170-induced mutations map at the other site containing threemonotonous G . C base pairs at position 50. Thus, ICR-170 appears to bepartially specific, causing mainly G .C insertions in contiguous G . C stretchesas well as lower frequencies of other types of alterations. However, we have

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ICR-170-INDUCEDMUTATIONS IN YEAST 237CYCl+ cyc 7-15

A C G T A C G T

-C

C

-- A- AA= G

-

TCCET

C

C

FIGURE . -Autoraa iog~am 01 a por t ion or t n e gel used to sequence the cycl-15 muta t ion . TheDNA sequences o f th e per t inen t cycl-15 a n d C Y C P eg ions o f the t r anscribed st r ands were de-termined with pr imer D, using the procedu res described by ERNSTet al . (1985) . T h e nuc leo tidesinvolved in the cycl-15 muta t ion and the normal r e la ted nuc leo tides o f t he CYCI+ s eq u ence a r eunder l ined .1 5 10 IS 20. .. ..(I l cL)Thr-Clu-Phe-Ly.-Al . -Gly-Ser-Al . -Lya-Lys-Cly-Ala-Th~-~-Ph~-Lya-Th~-A~g-Cy~-Le~-A X AC T C M T I C MG GCC GGT TCT GCT M G AM CCT W T CA CTT T SC M G ACT f f i A TCT CT A

805o'5

25 30 35 40 45 50EGlbCy~ -H im-~r -V~ l ~ l ~ -Ly~ -C~y -C l ~ -Pr~H i~ -Lya -Va l -G l y -Pr~ -A.~ -~~ -~ im -G l y - I le -Phe -G~y -Arg -H~a-Ser -G l y -G ln -A l~ -G lu -C~y -C M TCC CAC ACF Cn: GM A1m CGC CCA CAT M C ClT CC'f CCA M C 1TG CAI CGT AT C GCC ACA CAC ICI CCl C M C C I-5 90 105 li0 1;5 150

55 60 65 70 75 80~ r - S . r - ~ r - ~ h r - A . e A I ~ - A . b Ic L Y ~ - L y s - A . n - V a l - L c u - T r p - A . p - C l u - A . n - A . n y r - l le-TAT TCC TA C ACA CAT GCC M T ATC MG AM M C Cn: 1TG T U ; C A C G M M T C A X TCA C f f i TA C lTC ACT MEU M C AM TAT A T I165 I b O 145 z io 2;s 210

FIGURE . -The am ino acid sequen ce of i so - l cy tochrome c a long wi th the cor responding DNAsequences o f the nont ranscr ibed s t r and . T h e amino- te rmina l r es idue of meth ion ine tha t is excisedfrom th e ma ture protein is shown in parentheses . T h e am ino acid res idues are n u m b er ed ab o v ethe top rows, s tar t ing with t he am ino- terminal threonine. Similar ly, th e nucleot ides are n u m b er edbelow the b ot tom rows, s tar t ing with th e f i rst t rans lated nucleotide, A. T h e s i te of t h e cycl-15muta t ion a t amino ac id pos i tions 29-30 and the cycl-21I muta t ions at am ino acid pos i tions 75-76ar e doubly under l ined . Th e o th er four s i tes a l so hav ing GGG or CCC sequences are singly under-lined.not rigorously excluded that a small portion of the cycl mutations derived afterICR-I 70 treatments arose spontaneously. The preferential mutation at posi-tions 29-30 and the lower frequencies and lack of mutations of other sites,shown in Figure 3, clearly established that not all contiguous G - C stretchesare equally mutable.

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J. F. ERNST, D . M . H AM P SEY AND F . SH ERM AN

3. -T-G - -T - A - -G -G -T - G -T A T T-C-C-I 5 'I G T-9565 70 I \75- G GI \C T

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ICR-170-INDUCED M U T A T I O N S I N Y E A S T 239Although we have no explanation for the higher ICR-170-induced mutability

of the G -C stretch at positions 29-30, it should be noted that this site containstwo adjacent G .C base pairs, but in a different orientation (Figure 3). Althoughother mutagens have been observed to induce frameshift mutations, includingadditions and deletions of GSC base pairs (ERNST,STEWARTnd SHERMAN11982; STEWARTnd SHERMAN974), mutations at the cyc l -15 site were in-duced only with ICR-170 and not with any of the other mutagens listed in thelegend to Figure 1. By contrast, the Pro76 site, which includes only the singleICR-170-induced mutation cycl-211 (Figure 3), is highly mutable by U V . U Vwas shown to induce single and double base-pair substitutions, but not frame-shift mutations, at this site (Figure 1 and ERNST, TEWARTnd SHERMAN981,1982; ERNST t al. 1985).

The action of ICR-170 on the CYCZ locus appears similar to its action onthe HIS4 locus in yeast. Fine-structure mapping showed that eight out of 39ICR-170-induced mutations mapped at one site (CULBERTSONt al. 1977).Although the DNA sequence of this mutable site was not reported, DNAsequences of five different his4 mutations at other sites established that ICR-170 caused insertion of a G -C base pair. Three mutations occurred in runs oftwo G - C base pairs, and one mutation each occurred in runs of three andfour G.C base pairs (DONAHUE,ARABAUCHnd FINK 1981).

Also, the result of ICR-170-induced mutations in yeast is similar to that ofICR-191-induced mutations in E. coli. ICR-170 and ICR-191 are structurallyrelated, but for unknown reasons ICR-170 is more active in yeast, Neurosporaand Drosophila, while ICR-191 is more active in E. coli and Salmonella typhi-murium [see CREECHt al. (1972) for early references; CULBERTSONt al. 1977;F. SHERMAN,npublished results]. A comprehensive investigation of 373 ICR-191-induced mutations in the lacl gene, including DNA sequencing of 32mutations, established that nearly all mutations were additions or deletions ofsingle G. C base pairs at sites containing monotonous runs of three or four G.C base pairs (CALOS nd MILLER 1981). Also similar to the results observedwith the ICR-170-induced yeast mutations, certain sites in the ZacZ gene werehighly mutable with ICR-191. Both additions and deletions of G.C base pairsoccurred at most sites, while only (or almost only) additions of G -C base pairswere observed at other sites. In spite of the extensive results, the types ofnucleotides adjacent to the contiguous G-C stretches could not be simply re-lated to the susceptibility to ICR-191 or to the preference for additions ordeletions.

Even though additions and deletions of G -C base pairs have been observedFIGURE .-A suggested mechanism for ICR-170-induced G.C addition at nucleotide positions90-92 . T h e sequence from nucleotides 68 -90 would allow the formation of the indicated hairpinstructure with a AG = -15.5 kcal/mol (see Figure 3 for the complete nucleotide sequence of the

CYC l gene.). An insertion of G in the bottom strand, shown by the arrow, would allow furtherpairing at the base of the hairpin structure as indicated at the bottom of the figure. Return ofthis structure to the linear form and subsequent D N A replication or mismatch repair wouldaccount for the observed G . C addition at nucleotide position 90. ICR-170 may promote thisprocess by causing the formation o f looped-out structures shown in th e figure.

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240 J . F. ERNST, D. M. HAMPSEY AN D F. SHERMANin E. coli with ICR-19 1, while only additions have been observed in yeast withICR-170, it is unclear whether this difference reflects differences in mutagenicaction or whether only one type was uncovered in yeast because of the limitednumber of c y 1 mutations examined in our study and because of the limitednumber of highly mutable sites within the small C Y C l gene. Although some,but not all, ICR-170-induced mutants also can be reverted by ICR-170, thereverse mutations may not necessarily involve a deletion of a G.C base pair,but could involve external suppressors or other types of second-site alterations(CULBERTSONt al. 1977). At this time, w e believe it has not been excludedthat the action of ICR-170 in yeast is exactly the same as the action of ICR-191 in bacteria. Although the ICR-170-induced frameshift mutations in yeastmay arise by one of the general mechanisms proposed for frameshift mutagen-esis (see STREISINGERnd OWEN1985), there is still no adequate explanationfor preferential mutation at certain sites. However, a consideration of theformation of potential D N A secondary structures suggests a possible mecha-nism to account for the observed G . C base pair addition at nucleotide positions90-92 in the C Y C l gene (Figure 4; ee legend for details). Although formationand subsequent processing of this structure could account for the ICR-170-induced frameshift at this site-which is the predominant site of ICR-170action on the C Y C l gene-a similar mechanism cannot be invoked to explainthe ICR-170-induced lesions at the other sites in C Y C l (Table l ) , nor does itaccount for stretches of three G -C base pairs as the preferred site of ICR-170action.

This investigation was supported in part by the United States Public Health Service researchg r a n t R 0 1 GM 12702 and the N a tiona l Inst itu te s o f H ea l th t rain ing g ran t T 3 2 GM 07098 and inpart by the United Sta tes Departm ent of Energy grant DE-FG02-85ER60281. D.M.H. w as t h erecipient of an American Cancer Society Postdoctoral Fellowship PF-2347.

LITERATURE CITEDCALOS,M. P. and J. H. MILLER, 198 1CREECH,H. J., R. K . PRESTON,R. M. PECK,A. P . O'CONNEL L nd B. N . AMES, 19 72

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Proc. Natl. Acad. Sci. USA 7 8 6334-6338 .ERNST,J. F., J. W . STEWARTnd F . SHERMAN,982 Form ation of composite iso-cytochromes cby recombination between nonallelic genes of yeast. J . Mol. Biol. 161: 373-394.

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Communicating editor: I. HERSKOWITZ