Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light

J. Mol. Riol. (1984) 173, 273-291

Changes in DNA Base Sequence Induced by Targeted Mutagenesis of Lambda Phage by Ultraviolet Light RICHARD

I). Woon,

THOMAS

R. SKOPEK?

ASD FRANKLIX

HUTCHINSON

Department of Molecular Biophysics and Biochemi&y Yale University, New Haven, Corm. 06.511, I’.S.A. (Received

1 July

1983, and in revised forwl 31 October 1983)

Tn targeted mutagenesis of lambda phage by ultraviolet light, the mutations are caused by radiation-induced lesions in the phage DNA. Of 62 mutations in the lambda CI gene that were sequenced, 41 (63%) of the targeted mutations were transitions, with similar numbers of C. G to T. A and T. A to C. L’ base changes. The remaining 21 mutations were about equally divided among eight transversions, seven frameshifts (5 additions and 2 deletions), and six double events with either two nearby base changes or a base change and a nearby frameshift. Of the 62 mutations, 60 could be associated with -Pvr-Pyr- sequences in the DNA, sites of likely photoproducts. For more information on this point, lambda phage were irradiated with 313 nm light in the presence of acetophenone, for which the major photoproduct is reported to be the thymine-thymine cyclobutyl dimer, with no measurable Pyr(G-4)Pyo photoproducts. Of 22 mutations sequenced, 19 were transversions and only one was a transition. permitting the conclusion that thymine-thymine cyclobutyl dimers are not the primary cause of ultraviolet light-induced transitions. A consideration of all the data strongly suggests that Pyr(R-4)Pyo photoproducts are mutagenic lesions.

1. Introduction Mutagenesis by ultraviolet light has been extensively studied (for reviews, see: Hall & Mount, 1981; Witkin, 1976). Despite a wealth of detailed information. a coherent’ picture of the processes involved is only just starting to emerge. Tt is convenient to describe the processes in prokaryotes in terms of mutagenesis of lambda bacteriophage by ultraviolet light. For irradiated phage, a significant level of mutagenesis is usually seen only if the host cells are also irradiat’ed (Weigle, 1953). The effect of host cell irradiation is t’o derepress a set of genes concerned with DNA and DNA repair (Kenyon & Walker, 1980). Damage to the ~~11DNA activates the RecA protease, which cleaves the 1exA gene product (Litt~le & Mount, 1982) and allows the transcription of, among others, the urnuP gene (Bagg et al., 1981; more accurately the umuC and umuD genes, see Elledge & L%:alker. 1983). Only Escherichia coli cells wit,h urn& (and urn&) activity normally show mutagenesis by ultraviolet light (Kato & Shinoura, 1977: t Present address: Chemical Industry

lnstitutr of Toxicology.

Research Park. N.C. 27709. T.S.A.

273 (M~2~283ti/84/070273-19 IO

$03.00/O

63 1084 Academip

Press Inc. (London)

Ltd.

51

II.

I). \VOOl).

‘1‘. II. StiOl’EK

ASI)

I<‘. HI”l’(‘HISSOS

1981). It) is not kno\vll if protluc*ts Steinborn, 197X) or 1)~ X-rays (Kate & Nakano. of other genes under t,he wnt,rol of the f,exA repressor are also nr~rdrtl. This process is frequently referred t,o as targeted mutagenesis (iVitkin & iVermundsen. 1978) because of t,hr need for lesions in the gene in which thus phage. Mut’agenesis ot mutation occurs; in this case, a gene in the lambda bacterial cells by ultraviolet light is generally thought to depend both on phot’oproducts induced in the mutated gene and the levels of zrmu(’ and unlul) gene products (Hal1 & Mount, 1981; Little & Mount. 1982), but there is no direct’ proof. The existence of a different kind of mutagenesis, non-targeted mutagenesis, is shown by the high levels of mutation in non-irradiated lambda phage grown in heavily irradiated host cells (Uevoret, 1965; Tchikawa-Ryo & Kondo, 1975). In the accompanying paper (Wood & Hutchinson, 1984)) we show that non-targeted mutagenesis involves mechanisms quite different’ from those for targeted mutagenesis. It should be noted that in all experirnents on targeted mutagenesis, In experiments in which phage there will also be some non-targeted mutagenesis. and host cells are irradiat’ed separately, the level of non-targeted mutagenesis can be estimated from the results of similar experiments with non-irradiated phage. In this study of targeted mutagenesis, we determine the changes in DNA base sequences induced by ultraviolet irradiation of lambda phage. Mutants with the clear plaque phenotype arr isolated, and sorted into the ~1. cl1 and ~111 genes by complementation tests. Each c1 mutation is mapped by genetic crosses, the appropriate DNA restriction fragment is isolated. and then sequenced (Skopek & Hutchinson, 1982). The primary results obtained are the relative numbers of different kinds of mutations induced (transitions, transrersions, frameshifts, etc.). and the sites at which they occur. The present data are most directly comparable with those of 1,eClerc & Istock (1982), who sequenced forward mutations induced by ultraviolet light in the E. coli lac promoter cloned into the single-strand DI\‘A phage Ml3. The largest are those in which number of relevant papers in the literature, however, ultraviolet light is used to induce reversions of mutations in known DNA sequences (Lawrence 8 C’hristensen, 1979; Brandenburger ef al.. 1981: Kato & 1982) or forward mutations to amber or Nakano, 1981; Schaaper B Glickman, ochre codons in known sequences (Coulondre & Miller, 1977: Todd K: Glickman, 1982). Such studies are complement,ary t,o ours. in that the genetic methods provide data on a far larger number of mutations than are current’ly practicable by direct sequencing, but at the cost of restrictions on the kinds of mutations detected and the numbers of available sites.

2. Materials and Methods (a) Chemicah

and

rnsymes

Reagents were obtained from the following sources: cesium chloride, piperidine. formamide. deoxyribonuclease I and ribonuclease A from Sigma Chemical Co. (St Louis. MO); urea, phenol and restriction endonucleases HindIII. HhaI and BglIT from Bethesda Research Laborat,ories, Inc. (Gaithersburg, MD): acrylamide and bis-acrylamide from BioRad Laboratories (Richmond. CA): dimethylsulfate and a-aminoacetophenone from

TARGETED

MUTAGENESIS

BY CLTKAPIOLET

LIGHT

27,s

Aldrich Chemical Co., Inc. (Milwaukee, WI); hydrazine from Eastman Kodak Co. (Rochester. NY): E. coli DNA polymerase I from Boehringer-Mannheim (Indianapolis. IE); deoxyadenosine [32P]triphosphate (2000 to 3000 Ci/ mmol) from Amersham (Arlington Heights. TL); deoxyguanosine t.ripl1osphat.e from Calbiochem (La Jolla: CA). (b) Phage and host cell .sfrai~~s Mutations were induced in phage lambda ~1857 indl Oam, stored as a lysogen in E. coli Al5 SUP E44 host cells. Phage preparations used for exl)eriments were made by incubating a culture of the lysogens at, 5 x lO*/ml in a waterbath a,t 42°C for 15 min, then incubating for another 15 h at 37°C. Upon addition of a few drops of chloroform, a culture of 10” to 4x 10” plaque-forming units/ml was obtained. with less than 16” rlear plaque formers per plaque-forming unit. The low level of clear plaque mutants obtained by t’his I-st,ep growth procedure is essential for the experiments described in this paper. In the experiments to produce mutations by treatment of infectious lambda DNS followed bj transfection. DiVA was extracted from lambda ~1857 indl Onm. b615 b519 Tn2. prepared as described by heat induction of an Al5 lvsogen. The host cells used in these experimrnt,s were AKIN86 F- wrA6 nrg his IPU pro thr m-n gnl Inr ntfl x,1/1thi. (c) Media

and plates

Cells were grown in K medium, which consisted of (per liter): 1 g KH,Cl, 5.9 g NaHPO,, 3 g KH,PO,, 0.25 g MgSOk.7H,0, 11 mg CaCI,. 0.1 rng thiatnine, a,nd 1% (n/v) glucose, la& (w/v) Casamino acids (decolorized). The medium in lambda plates contained (per liter): 1 g XaCl, 5 g Bacto Tryptone, 8 g Bacto Peptone and 15 g Bacto agar. Top agar contained (per liter): 8 g NaCl, 5 g Bacto Tryptone. 5 g Bacto Peptone, and 6 g Bacto agar. Phagc were diluted in buffer containing 6 rn>f-Tris. HCl (pH 7.2) and 2.6 g M&W,. 7H,O/l. (d) Mutagenesis

by ultm&let

light

The phage were irradiated in suspension at about 5 x lo* p.f.u.t/ml in lambda buffer with a germicidal ultraviolet lamp (maximum output at 254 nm) at’ 1.0 J mm2 s-i. For sensitized irradiation, the phage (or infectious DNA) were suspended in 4 mM-or-aminoacetophenone hydrochloride (Lamola & Yamane, 1967: Rahn, 1979)> at 6x IO9 p.f.u./ml in a pH 7.0 buffer containing 13 m,ur-phosphate. 3 mM-MgSO, and preincubated for 16 h at 4°C. Samples were irradiated with light from a water-cooled, medium-pressure mercury discharge passing through 2 cutoff filters (Corning O-53 and Corning 7-54) and an interference filter (Ealing Corp., S. Katick. MA) with peak transmission at 313 nm. The flux as measured by ferrioxalate dosimetry (Calvert & Pitts. 1966) was about lo3 J m-’ min- ‘. A complete description of the light source is given elsewhere (Krasin & Hutchinson, 1978). All irradiations with 313 nm light were carried out in the absence of oxygen gently bubbling nitrogen through the solutions. The mutagenic procedure was different for each of 3 types of experiments, and the d&ails are given in footnotes to Tables 1 and 7. (t‘) Mappin,g

of mutations

,Mutant phage were obtained from clear plaques chosen at random, and a complementation spot. test (Belfort et al.. 1975) was used t,o distinguish cI mutants from ~11 and cITT mutants. t Abbreviation usrd: p.f.u., plaque-forming units

Mutated base x0.t - 69 -H-(-65) - 40- ( - 39) - 33 3 1%20 24 3I 40 43 55 55 58 59 71-77 7x 79 79-83 95 124

152 154 157 160 160-161 166 166 169 173 176 208 214-215

221 223 223 231 235 Ir 236 239 251

3 17-322 410-41 I 499-502 545 tiO7~6lI

61 1

(‘hange: (1 + ‘I’ -(’ (K: -+ TT (:+T (‘ --t T + I’yr T-A (‘+T (‘+I (:+T (‘-+ T (’ -9 I’ur T-+(’ T-C’ + Pyr (‘+TT7 ‘I-+(’ +Tl T-C’ (!+‘I T-c: T+A ‘I‘+(’ (: + I C(: + T’J T-C’ T + (: (‘+T ‘r + (‘ 1’ + A (‘-bT (:A -+ TT T + (’ (‘-+A (!+‘IT + A c: -+ 1 (‘-+T (I + ‘I T-C‘ +T (X1-*

(Change Op.-promoter Op.-promoter op. promoter Op.-promoter Met -+ Ilr I ,eu -+ l’hr (:lu + Lys (:lu -+ Lys Asp + Asn I ,eu --+ Phr I ,PU + ! Lys + (:lu Lys + Arp

0 7 10 I3 14 18 18 I9 I9

2 2

3 I

4 2

Lys + Clu

26

Leu + SW My + Arg Leu + Ser Phr -+ Ile Asn + Asp (:ly + Srr (:Iy --t Asn As11--a Asp Asn + His Ala -+ Thr I,eu + Ser AH1 -+ Tie Leu -+ I’he Val + Asn \‘a1 + Ala (:lu + llAA (:lu -+ Lys Phe -+ Leu Pro + Ser Pro + Lru SW + Leu (:1u + Gly

31 41 50 51 52 53 53 55 .5.5 56 57 5x 69

4

2

2 I

71 73 74 76 76 78 78 79 83

A

GAAT + AAA T + A - ‘, T-C’

I Ic + Lys Leu --t ser

181 203

Lambda ~I857 indl Oclm phage were irradiated with 30 tJ rn-’ of 254 nm light and adsorbed to AU1886 uvrA6 host cells (given 3 .J m -*) at a multiplicity of infection of less than 0.1. The culture was incubated for 2.5 h to allow the host cells to burst, chloroform was added, and total plaque-forming units and clear mutants were determined. Plaque-forming ability was 6% of that of non-irradiated phage. The induced mutation rate was 460x 10m5 clear mutants/p.f.u., compared to 9x 10e5 clear mutants/p.f.u. for non-irradiated phage assayed in irradiated host cells. (With uvr- umuC36 host cells

‘I’ARCETEI)

M1’TAC:ICSESIS

BY

I’I,THAVIOLET

LIGHT

277

The CT mutants were mapped into the following regions of the c1 gene: operatorpromoter: base-pairs 1 to 125; base-pairs 125 to 240: base-pairs 240 to 352: base-pairs 352 t,o 47X; base-pairs 478 to 714. The base-pair numbers are those given by Sauer (1978). This was done by genetic crosses wit’h phages previously described (Skopek & Hutchinson. 1982). which have various parts of the CI gene delet,ed.

Most mutants chosen for sequencing were a random selection of those with mutations between base-pairs 1 and 240 (see Table 2). A proport,ionate number mapping in the oI)~‘rator-I’romoter region were also sequenced. A smaller fraction of those mapping I)rt\vwn base-pairs 241 and 714 were sequenced because fewer of these were base substitutions, which in this work gave the most information about mutagenic mechanisms. The sequencing of mutants was carried out as described (Skopek & Hutchinson, 1982). Briefly. IO to 15 pg of lambda Dlu‘A were digested either with a mixture of Hind111 and NhoT restriction endonucleases. or with RglIT endonuclrase. depending on the mapped location of the mutation. In the first case, the recessed 3’ ends of the Hind111 cuts were labeled with [cc-32P]dATP, using /C. coli DKA polymerase I at 0°C’: no labeling of the HhnT c,uts (*an o(‘cur under these conditions. The appropriate restriction fragment. labeled at one end. was then isolated by electrophomsis on a 5.5% (w/v) acrylamide gel. In the second (Base.the 3’ end in the recessed BglIT cut was labeled with [cr-32P]dATP in the presence of non-radioactive dQTP, again using E. coli DEA polymerase I at 0°C’. The DNA was then digested with HindTTI, and the appropriate restriction fragment. labeled only at the BgZII taut. isolated on an acrylamide gel. In either case. the desired band was cut out of the gel. the restriction fragment was elutrd. and then sequenced by the method of Maxam & Gilbert (1977). mapping

3. Results For t#his study of targeted mutagenesis by ultraviolet light. the host cells chosen werr z/w - and lacked the ability to excise cyclobutyl pyrimidine dimers (and other lesions) from DNA. This provides a somewhat simpler situation t)han that for II/‘~ + cells, where an unknown number of the lesions induced by ultraviolet light will be excised before a mutagenic event takes place. The host cells were irradiated with 3 J mm2 of 254 nm light. t’hr lowest exposure that fully induces the process of Weigle mutagenesis for infecting lambda phage. Thr lambda ~1857 indl Oam phage were irradiated with 80 J mm2 of 254 nm light, and adsorbed to host cells. After the burst. the frequency of clear mutants per p.f’.u. was about X-fold higher than the rate for non-irradiated phage assayed in irra,diated host cells (footnote, Table 1). Thus. virtually all the mutants selected for sequencing were those induced as a direct result of lesions in the phage gfmmt’,

given 3 .I m -‘, irradiated phage gave 1 x 1OF5 clear mutants/p.f.u.) The 40.ml culture contained a total of 1360 independent bursts of mutant phage; statistical calculations show that, with 98y; probability, at least 58 of the 62 mutants sequenced were from independent mutational events. Of 150 (-Iear mutants: 3Q (26%) were mutations in ~11 or ~111, as determined by complementation tests; of the II 1 ~1 mutants, 98 were mapped (Table 2), 4 gave inadequate mapping data (low yields of recombinant phagr, contaminated plates, etc.), 2 gave confusing mapping data that were never resolved, and 7 were not investigated. t Base numbers are from Sauer (1978). : Thr pyrimidinr of the mutated base-pair is given first. All sequences of 2 or more bases are given 5’ + 3’.

IZ I). L\‘OOI).

‘1’. I<. SKOl’Eli

XSI)

IJ. HI”I’(‘HtNSOS

TAFII~E 2 Spectrwn

of mutcltims

induced

~~mrnary

by ultraviolet

qf the

light

rmult
ix the cl grnr

in Table

in i. clX5T.~

1

Region of cl gene

(‘orrected operatorpromoter Mutants mapped Mutants sequenced Types of mutations Transitions: C C: --t T A T.A+C.G Transversions: Prameshifts: - 1 base-pair + 1 base-pair + 2 base-pairs Double events: 2 base changes 1 base change and frameshift

Base-pairs

Base-pairs

l-240

241-714

19 7

8

71

5

50

3

20

0 0

16

Totals 98 62 41

7

0 2 1

1 0 0

0 3 1

1 1 0

1

2

0

0

1

2

fraction of totalt

0.63 0. I3

8 7

0.13

6

0.1 1

t DiKerent fractions of mutants that map in various parts of the gene were sequenced, as shown in the t)op 2 entries in the Table. The calculation of the best estimate for the occurrence of a particular type of mutation can be shown by an example. Transitions are 36/50 = 0.72 of the mutations sequenced in the region base-pairs 1 to 240, which comprise 71/98 = 0.725 of the mutations mapped. Thus, transitions between base-pairs 1 and 240 are 0.72 x 0.725 = 0.522 of the mapped mutations. Similar calculations give (3/5) x (S/89) = 0.049 and (2/7) x (19/98) = 0.055 for the fraction of mutations that are transitions in the operator-promoter region and between base-pairs 241 and 714, respectively. Thus, the best estimate for the fraction of transitions among the mutations is 0.522 fO.049 f0.055 = 0.626. The fractions of other types of mutations are calculated in a similar way.

The changes in base sequence for 62 mutations are listed in detail in Table 1 and in summary form in Table 2. About two-thirds were transitions, equally divided between C. C to T. A and T. A to C. G. The remainder were about equally divided among transversions, frameshifts and double mutation events. The last category includes mutations in which two base-pairs were changed, or in which both a base change and a frameshift occurred. Tt is reasonable to ask if the sites of these targeted mutations are correlated with the sites of the lesions in the phage DNA that induce the mutations. Such correlations have been suggested (Brash & Haseltine. 1982; Miller, 1982: Foster Pt al.,

1982).

The most frequent photoproduct induced by 254 nm light is the pyrimidine cyclobutane dimer Pyr = Pyr, and the next most frequent is the Pyr(6-4)Pyo photoproduct (Patrick & Rahn, 1976; Brash & Haseltine, 1982); the structures are shown in Figure 1. In double-helical DNA, either photoproduct can occur only at adjacent pyrimidines on the same strand. Thus, a pyrimidine in a sequence -I’m-Pyr-Purcannot be part of either product. Of the 62 mutations induced by ultraviolet light (Table l), only two have the pyrimidine of the affected base-pair located in such a sequence; this strongly suggests that the sites of mutation are

TARGETED

MUTAGENESIS

RY ITLTRAVTOLF,T

LIQHT

279

Fro. 1. The structures of the pyrimidine-pyrimidine cyclobutyl dimers (Pyr = Pyr) and the Pyr(664)Pyo photoproducts induced in DNA by ultraviolet light (Cohn et al., 1974). The mutagenic Pyr(6-4)Pyo lesion could be the form on the left with the 4-member ring, the more stable form on the right (Haseltine, 1983), or even the form found after acid hydrolysis, in which the -YH group (Y is -NH- for cytosine or 0 for thymine) has been lost and the pyrimidine ring on the 5’ side has reacquired the 5-6 double bond.

correlated with the sites of photoproduct’s. Tnformation on sequence specificity is summarized in tabular form for transitions and transversions (Table 3): frameshifts (Table Ti), and double events (Table 6). The analysis of transitions and transversions in Table 3 is confined to sites hetween base-pairs 1 and 240. Most such mutations occur in t,his region, as may be seen by examining Tables 1 and 2. This is not just a characteristic of mutations induced by ultraviolet light,, but is a feature of mutations from several mutagenic agents (Table 4). Thus, a change in the first 80 amino acids (coded for by basepairs 1 to 240) is tenfold more likely to affect repressor function than a change in the remainder of the polypeptide (see also Skopek & Hutchinson, 1982). This is a consequence of the structure of the repressor protein (Pabo et al., 1979; Sauer CJ~ al.. 1979). The amino terminus forms a domain that recognizes the operator sequence in DNA, and its function is therefore likely to be affected by a large number of amino acid changes. The chief function of the carboxy terminus seems t’o be association with the carboxy domain of another repressor molecule to form a dimer, a function that might reasonably be less sensitive to changes in amino acids. Note t,hat those mutations that cause more drastic changes in the polypeptide, frameshifts, large insertions or stop codons. are more evenly distributed throughout the gene (Table 4). The small number of transitions at Pyr* in the sequence 5’-Pur-Pyr*-Pyr(Table 3) shows that either: (I ) two Pyr groups preceded by a Pur rarely form a photoproduct; or (2) transitions occur selectively at the 3’ end of photoproducts involving two adjacent pyrimidines.

‘!hnsitiorrs

Tranhversions

Sitrs mutatedt

Sitrs mut,atedt Sites3

By all agents

By U.V.

No. of u.v. mutants

33 40 x5

9 I3 29

1 1 20

158

51

T-f’* (‘.(‘* T.‘r* (‘-T*

I6 I5 38 I6

Totals

85

SO. of u.v. mutants

Sites3

By all agents

10 U.V.

1 I 34

47 rI I&

8 8 20

0 2 5

0 2 -5

22

36

206

30

7

7

8 8 9 4

7 5 8 0

11 8 15 0

29

20

34

‘ I 3

5’

Pur-Pyr*-Pur Pur-pyr*-Pyr Pyr-pyr* Totals

t The number of sites at which a transition (or transversion) has been shown to cause loss of repressor protein function. The columns headed all agents include mutant sites of spontaneous origin, mutations induced by bromouracil and ultraviolet light (u.v.), and 19 mutations of various origins for which the sequence changes are given by Hecht et al. (1983). $ The asterisk (*) indicates the pyrimidine in the mutated base-pair. SThe number of sites between base-pairs 1 and 240 in the c1 gene at which a transition (or t,ransversion) changes the amino acid.

Data on the relative yields of Pyr = Pyr and Pyr(Cj-4)C photoproducts at various sites in a portion of the E. coli lacl gene have been published by Brash & Haseltine (1982); in addition, Figure 2 gives some qualitative data on such yields for a 92-base sequence in the lambda c1 gene. Both the results of Brash & TABLE 4

The number of mutated sites in parts of the lambda cl gene for 288 sequenced mutations that were induced by various agents Number of mutated sites

Type of mutations Missense (change in amino acid) Frameshifts, large insertions (18 elements), nonsense (stop codons)?

Uase-pairs I to 240

Base-pairs 241 to 714

00

7

I6

I6

Mutations of spontaneous origin, and those induced by ultraviolet light, bromouracil. 9-aminoacridine, proflavin and ICR-191. t For various reasons, the fraction of mutations selected for sequencing is lower for those mapping in the carboxy terminus than for those in the amino terminus (e.g. see Table 2). The true incidence of frameshifts, nonsense mutations and insertions may well be roughly proportional to the number of base-pairs in the DNA segment.

TARGETED

MUTAGENESIS

BY VLTRAVIGLET

LIGHT

2x1

TABLE 5

Frameshift mutations induced by ultraviolet light and by other mutagenic agents at various YWZSof identical base-pairs in the lambda cl gene

5 No. of runs in cl gene

1

2.54 nm light 313 nm and acetophenone Son-targeted mutagenesis by 1J.V.f Acridines$

No. of adjacent Q C pairs 4 3 2

3

-t 1 -1 -t 1 - 1

10

43 1 1

1

7

6

188

1

3

2

I

1

1

No. of adjacent A T pairs 5 4 3

3

0

18

2

1

Totals

70

159 5 2 2 I

1t

1

1

1

+ 1 -- 1 +1 --1

1 12 17

3 9 36

1

I I

I 2 1

t This is a f2 frameshift. $ Data from Wood & Hutchinson (1983): u.v., ultraviolet 5 Data from Gkopek & Hutchinson (unpublished results).

1

1

1

2 8 25 54

light

Haseltine and those presented in Figure 2 show that, in a sequence of three or more adjacent pyrimidines, the probability for the formation of Pyr = Pyr dimers is greatest for the pyrimidine pair at the 3’ end, and least for the pair at the 5’ end. The Pyr = Pyr yield in Pur-Pyr-Pyr-Pur sequences, however, is comparable TABLE 6

Double mutation events induced by ultraviolet light in the cl gene Hase changes only

Frameshift

- 40t -(:-C-G- -C -A-GP

A-A-A-A-A-A-A-A-

-A-AT-

G-G -C-A-TP -m214

-A-A-A-

plus base change 78 -A-A-A-A-A

-A-A- G-C -C-A-G P -a499 -T-T-F-T-T-P-T-

All sequences are given 5’ to 3’. The upper sequences are those for the wild-type non-transcribed (sense) strand. The corresponding sequence in the mutant is below. t The double mutations at base-pairs -40 and 160 actually represent identical events: T-G-G-C T-A-A-C A-C-C-G + A-T-T-G

‘XL’

Ii

Base no.

I). \VOOI).

‘I’, I<. SKOI’KK

ASI)

I’. Hl:‘l’(‘HlXSOS

160

I50

170

5' -G-C-i-T-T-A-T-i-T-A-A-T-~-G-C-A-T-~-A-A-Tn ,T h A

Sequelw breaks (A)

254 nm + endo 313 “m +endo 254 nm + olhali No. of muiants, 254

30 J m-‘,

Sequence breaks (A) 254 nm +endo 313 nm + endo 254 nm + alkali No. of mutants

w 5

m 5

m

m

s

G-c-A-T-T-A-i-A-T-Gh I\ w 5 5

A

5 s

I

I

190 180 -C-i-T-A-T-A-i\-C-G-C-C-~-C-A-T-T-i;-C-T-T-A-~-A-A-A-A-~-T-T-C-T-~-

(II

^,

n w

z s

h m m

I\

210

200 AA mm 5

I\ s 5

h m

A

w

s I

220 Sequence breaks (~1 254 m-n+ endo 313 nm + endo 254 nm + olkoli No. of mutants

2

(2)

(2)

(II

nm

230

240 3'

-A-A-A-G-i-T-A-G-C-c-T-T-c-n-i\-c-n-n-T-~-T-A-G-C-~-C-T-T-C-~A m “S (I)

rl Ill m

h Ill s I

(2)

h 5 “5

I\AAAII ww

I

h SIT 5

2w2w

2s

in base-pairs 148 to 240 of the non-transcribed strand of the FK;. 2. The location of photoproducts lambda rl gene as determined by the methods of Brash & Haseltine (1982). Lambda DNA was digested simultaneously with Hind111 and HhaI endonucleases and labeled with 32P ab the IiindilI 3’ ends as for sequencing. The restriction fragment containing base-pairs I to 352 of the c1 gene was isolated on a gel, as for sequencing (see Materials and Methods), and irradiated with 1000 J m-’ of 254 nm light or 2.5 x lo4 J me2 of 313 nm light in the presence of 4 mh%-a-aminoacetophenone. To locate Pyr = Pyr cyclobutane dimers, the irradiated DNA was treated with an excess of T4 endonuclease (30 min at 37°C in 100 mM-NaCl, 5 mM-EDTA, 10 mix-Tris. HCI, pH SO), which cuts DNA 5’ to a Pyr = Pyr dimer. The DNA was then electrophoresed on a sequencing-type gel; 2 adjacent tracks were used for the same restriction fragment given the standard Maxam-Gilbert. (A+G) and (T+C) treatments, so that the exact points at which the T4 endonuclease cut the USA could be determined. Pyr(6-4)C photoproducts were determined by treating irradiated DNA with hot alkali (1 M-piperidine at 80°C for 15 min) to induce breaks 3’ to the (6-4) products as previously described (Lippke et al., 1981). The strength of each band on the photographic film was estimated by rye: w, weak; m, medium; s, strong; vs, very strong. From comparisons with a set of standards having known amounts of 32P in each spot, the categories are roughly a factor of 2 apart. The way t,he experiments were done, no inferences ca.n be drawn concerning the numbers of lesions induced by any one treatment (e.g. 254 nm light + T4 endonuclease) relative to those induced by another treatment. The number of mutants without parentheses are those that may be associated with a -Pyr-Pyrsequence in the given sense (non-transcribed) strand; those in parentheses are assumed to be associated with a -Pyr-Pyr- sequence in the complementary (transcribed) strand.

to the average yield in sequences of three or more pyrimidines. Thus: the data of Brash & Haseltine show a yield of Pyr = Pyr dimers at Pm-Pyr*-Pyr* sequences that is about half that at Pyr-Pyr*-Pyr*. For Pyr(6-4)C products, the data are less extensive, but suggest a similar photoproduct yield at a particular Pyr-Pyr sequence whether preceded by a purine or a pyrimidine. The conclusion is that the low yield of transitions at Pyr* in a 5’-Pur-Pyr*-Pyrsequence cannot be explained by a low yield of photoproducts. Therefore, transitions must occur most commonly at the 3’ end of a photoproduet. Direct information on which photoproduct is mutagenic, Pyr = Pyr or Pyr(6-4)Pyo, was obtained by irradiating lambda phage with 313 nm light in the presence of the sensitizer a-aminoacetophenone (Lamola & Yamane, 1967: Rahn

TARGETED

MUTAGENES18 UY ULTRAVIOLE'I'

LIGHT

283

& Patrick, 1976; Rahn, 1979). Radiation of this wavelength forms excited triplet states in acetophenone, which can transfer energy to the triplet state of thymine but’ not to any other base. This leads to the formation of T = T and a low percentage of T = C cyclobutyl dimers (Rahn $ Patrick, 1976; Lin & HowardFlanders, 1976). No Pyr(G-4)Pyo photoproduct has been detected (Rahn $ Patrick, 1976: Rahn, 1979). The clear plaque mutants from sensitized irradiation (Table 7) show only one transition of 22 mutants sequenced. The results in Figure 2 show in a qualitative way that cyclobutyl thymine dimers are formed in the DNA irradiated under our conditions. Calculations based on the data of others show comparable numbers of dimers in lambda DiYA irradiated with 254 nm light and with 313 nm light plus acetophenone, at comparable levels of plaque-forming ability (Table 8). Since> mutations per plaque-forming unit are somewhat lower with 313 nm light plus acetophenone, the low fraction of transitions is not the result of a new and highly mutagenic lesion induced by sensitized irradiation that overwhelms transitions induced by cyclobutyl dimers. Tt may be concluded that T = T cyclobutyl pyrimidine dimers, at least, do not induce transitions efficiently. For DNA irradiated with 254 nm light. a weak band after treatment with phagr T4 endonuclease (Fig. 2) corresponds to a break between bases 169 and 170, which is not at the 5’ side of a pyrimidine pair. This break may represent an unidentified photoproduct’: it is interest,ing that a transition at base-pair 169 (Table 2) is one of the only two mutations induced by 254 nm light that are not associated with a Pyr-Pyr sequence.

4. Discussion Table 2 gives an estimate of the proportion of various types of mutations induced by 254 nm ultraviolet light in the c1 (repressor) gene of lambda phage: 63% transitions, divided about evenly between C. G to T * A and T. A to C. G; 10 to 1,504 each of transversions, of frameshifts, and of double mutations in which there are two nearby events, either two base changes or a frameshift and a base change. Of the commonly recognized types of mutations, only insertions or deletions of three or more bases were not found. One reason might be that our procedures tend to select against such mutations. Six of the CI mutants isolated gave ambiguous mapping data and are not included in the 98 listed in Table 2. Sequencing was begun on ten mutants but not completed for various reasons. A review of these cases produced no obvious reason why any type of mutation should have been discriminated for or against. Thus, the statistical upper limit for the number of insertions/deletions is 3.7 at the 95% confidence level, and this class of mutations represents less than 3.7162, or 6%, of the total. Note that insertions of 5000 or more base-pairs or deletions larger than 3000 base-pairs with a terminus in cI would produce non-viable phage. Conceivably, deletions (and insertions) were not induced in these experiments because they might require recombinational events bet,ween homologous DNA molecules, which could occur only infrequently at the multiplicity of infection used (0.1).

Mutated base ( ‘hange A. Irradiation 6 918 I5 36 97 I27 I’37 11 I’%‘, 1< 160 Ii!) 6X6

s0.t

of intact phug~ $11 (‘+ .4 + ‘r (1-A (:+A 1’ + PO 1’-t(’ (‘+A (‘+.4 (‘-A (‘+ A (’ + A

11. /m?&iation of lamhdn Il.\“4 (I 71 15 (:+A 71 -77 +T 76 T -+ c: 123 -(’ I3I c --+ c: I95 (‘ -+ A 260 (‘+ A 290 (‘+ c: 312 (’ + A 565 (‘ + A

Srr + Arg

I

1,~s + Asn (iIn -+ His ‘i,rr+! Met -+ Val (:I!1 + Lvs Gin + His my * (ys Ala + Asp Srr --t He

4 I1 32 42 44 44 53 59 228

Lys -+ Am

4

Lys + (:lrr

“5

I

1 2

1 Glv + ,4la Leu + Phe Cl” + I’AA Ser -i ITG.4 Tyr + I:AA (:I” -+ VAC:

43 64 83 96 I03 188

I

t base numbers are from Sauer (1978). $ The pyrimidine of the mutated base-pair is given first. 4 Lambda ~1857 indl Oam phage were suspended in a solut,ion of 4 miwa-aminoacetophenone and irradiated with 1150 J m-* of 313 nm light under anoxic conditions. Plaque-forming ability was 360, of that for non-irradiated phage; acetophenone without light had no effect. The phage were adsorbed at a multiplicity of infection of 0.1 or less to AU1886 uvrA6 host cells (given 3 J m-* of 254 nm light), t,he complexes incubated for 2 h at 37”(:, and products of the burst spread on lambda plates with Al5 lawn cells. There were 1328 bursts giving mutant phage in the culture, and a 940/,, probability that each mutant sequenced came from a different mutational event. For phage treated with acetophenone but not irradiated, there were 2.3 x 10m5 clear mutants/p.f.u.; for phage irradiated in the absence of acetophenone, there was a similarly low level of mutants; for phage irradiated in the presence of acetophenone, there were 64 x 10e5 clear mutants/p.f.u. In all, 42 mutations were mapped: 2 in the operator-promoter region, 33 between base-pairs 1 and 240, and 7 between base-pairs 241 and 714. 11Purified infectious DNA from lambda ~18.57 indl Oam 5515 b519 Tn2 phage at 10 pg/ml in 0.1 M-‘Iris (pH 7.6) with 4 mM-cc-aminoacetophenone was irradiated under anoxic conditions with 31OJ mm2 of 313 nm light. The DNA was used to transfect AU1886 WT- cells made partially permeable by calcium t.reatment (Davis e:1al., 1980). irradiated at 5 x lOs/ml with 3 J me2 of 254 nm light then concentrated to 10’O/ml. In this case, a mal mutant of the host cells, unable to adsorb lambda phage, was used to avoid readsorption of phage released in a burst. The culture was divided into a number of small tubes, which were diluted to 5 x 10s cells/ml, then incubated for I h at 37Y’. The contents of each tube was spread on a lambda plate with A15 lawn cells; only one clear mutant from each plate was selected, so each mutant is known to be of independent origin. Transfection efirirncy was 104 to IO5 plaques/pg irradiated DNA. DNA treated with acetophenone but not irradiated showed 3 x 10e5 clear mutants/p.f.u.; DNA irradiated in the absence of acetophenone showed a comparably low level of clear mutants/p.f.u.: DNA irradiated in the presence of aretophenone showed 180x lO-5 clear mutants/p.f.u. In all. 16 mutations were mapped: 1 in the

TARGETED

MUTAGENESIS

KY

ULTRAVIOLET

“85

LIGHT

TABLE 8

Comparison of the numbers of mutations and of cyclobutyl dimers induced by ultraviolet light between base-pairs 1 to 240 of the lambda cl gene In base-pairs l-240 Irradiation Irradiated form

Plaqueforming ability PO)

Clear

-

mutants

per p.f.u.t (x 103)

Mutations: ( x 103)

(nm)

(J m-‘)

Phagr

254

30

6

4.6

2.5

l’hage l)NA

31311 31311

1150 310

36 8

0.64 1.8

0.31 0.65

Cydobutane dimers (calculated)4 (x 103) 70 64 117 43 84

(1) (2) (3) (I) (4)

t In all cases the phage (or DNA) were assayed in AR1886 uv7A6 host cells given 3 J me2 of 254 nm light. Mutation frequency data are from the footnotes to Tables 1 and 7. Plaque-forming ability is given relative to unirradiated controls (phage or DNA). 1 This column gives the number of clear mutants in the region base-pairs 1 to 240, per total number of plaque-forming units, calculated as (total clear mutants/p.f.u.) x (fraction of clear mutants in ~1) x (fraction of cI mutants that map between base-pairs 1 and 240). 5 The numbers of T = T and C = T dimers are calculated for base-pairs 1 to 240 in the c1 gene from data in the references given below. Xote that C = C dimers are ignored; they are present in smaller numbers than the other two for 254 nm (Patrick & Rahn, 1976), and not formed by sensitized 313 nm irradiation (Kahn & Patrick, 1976). The irradiations (columns 2 and 3) used the same 313 nm light source and dosimetric procedure as in Lin & Howard-Flanders (1976). and the same dosimeter for 154 nm irradiation. References: (1) Lin & Howard-Flanders (1976); (2) Patrick (1977); (3) Garces & 1)avila (1982); (4) Rahn & Patrick (1976). 11Irradiation wit.h 313 nm light was in the presence of acetophenone.

Livneh (1983) reported that for a DNA fragment irradiated with ultraviolet light and then spliced into a plasmid, three of eight mutations were short, deletions near the ends of the fragment; it is likely that these deletions arose during splicing. (‘oncerning the data presented in Tables 1 and 2, the following points need t,o be emphasized. (1) These mutations were nearly all induced as a consequence of photoproducts in the phage genome. The increase in mutation frequency for irradiated phage versus non-irradiated phage in identically induced host cells was greater than Z-fold (see footnote to Table 1). (2) The relative numbers of kinds of mutations will depend on the gene product. For example, the large number of transitions in the CTgene from ultraviolet light (Table 2) comes from the sensitivity of the amino t,erminus to changes in amino operat,or-promoter region, 10 between base-pairs 1 and 240, and ;5 between base-pairs 241 and 714. * Two different irradiation conditions were used to minimize the chance of drawing incorrect conclusions because of certain complications. Irradiation of the phage with 313 nm light in the presence of acetophenone might cause crosslinks between the DNA and proteins or polyamines (Shetlar. 1980), which would not have been detected in previous measurements of photoproducts (Kahn & Patrick, 1976). Such crosslinks cannot be formed in significant numben in the experiments with isolated DNA. In the experiments with isolated DNA, the host cells were treated with calcium to permit transfection; unpublished data (this laboratory) suggest that induction of Weigle mutagenesis hy ultraviolet radiation of permeabilized cells may be different from that for normal cells.

It.

2Mi

I). \VOOI).

‘I’

It

StiOl’EK

ASI)

I;. H\.‘l’(‘HINSOS

A gene product whose sensitivity to changers in a,mino acaid \~as mot’(’ likt, that, for ba,se-pairs 241 to 714 would show a higher fraction of framrshitf mutations. (3) These results are for uvr cells, and the lack of this excision activity can affect mutagenesis in at least two ways. (a) The effective distribution of mutagenic lesions in the DNA can change because of differential excision of photoproducts ill IL’W+ cells. (b) There may be a major difference in mutational mechanisms in excision-proficient and excision-deficient strains. For example, the first replication of each genome after mutagenesis of yeast cells by ult8raviolet light, yields two mutant genomes in excision-proficient cells. but only one in excision-deficient cells (James & Kilbey, 1977; Eckhardt et al., 1980). Analogous result,s for E. coli are less clear-cut, but suggest a corresponding difference between uwf and UZY- cells (Nishioka & Doudney, 1969,197O). The spectrum of mutations induced by ultraviolet light in lambda phage is in good agreement with that obtained by LeClerc & Istock (1982) for the E. coli lac promoter cloned in the single-&and phage Ml3 (Table 9). While t’hey used UI;T+ cells, it is likely that the excision enzymes would not act on single-stranded Ml3 DXA, and thus their results would be more directly comparable t’o t’hose involving double-stranded DIVA in UVT-. rather than uvr + cells. witls.

(a) Transitions Coulondre & Miller (1977) characterized a large number of mutations induced by ultraviolet light that gave amber or ochre stop codons in the E. coli lacl gene on a plasmid in uvr+ host cells; similar data for the la& gene in UVT+ and uvrcells have been given by Todd & Glickman (1982). These results for the Zacl gene TABLE 9 Targeted ultraviolet mutagenesis of E. coli lac promoter cloned in Ml3 stranded DNA phage (LeClerc & Istock, 1982) Number of mutations Mutation Transitions (‘+T T + (1 A-G Transversions Frameshifts Double evental

Pur-Pyr*-Pyr

Pyr-PyP

at locations:t Pur-pyr*-Pur

8 16

0 I

I 2

4

0

0

PUr*

2 2

single-

0

1 0

Totals

9 19 2 ,5 2 3

Mp2 (Ml3-lac hybrid) phage were irradiated with 125 J m-* of 254 nm light, and grown in UW+ recA + lexA+ host cells irradiated with 50 J m-‘. The induced mutation rate was 12 times the rate for non-irradiated phage grown in either non-irradiated or irradiated host cells. t The asterisk indicates the mutated base-pair. $ Double events: 5’.C-C-C-A-G- --+ -C-C-A-C-T-T-T-A- --t -T-T-A-T-G-T-A-T-+-G-A-C-T-

TARGETED

MUTAGENESIS

BY

ULTRAVIOLET

LIGHT

285

are presented in Table 10 in the same form as ours in Table 3 for the lambda c1 gene in uvr- cells. A number of conclusions may be drawn about. transitions induced by ultraviolet light. (1) Transitions are strongly correlated with -Pyr-Pyr- sequences and occur with low frequency at Pyr* in -Pur-Pyr*-Pursequences (Tables 3 and 10). This conclusion is reinforced by the unequivocal location of 25 of 30 transitions in single-stranded DNA at -Pyr-Pyr- sites (Table 9); for double-stranded DNA, one cannot distinguish between mutations at -Pyr-Pyr- sites and those at purines in the complementary -Pur-Pur- sequences. (2) Transitions occur preferentially at the 3’ end of a Pyr-Pyr sequence: see Results and Table 9. (3) T. A to C. G and C. G to T. A transitions are induced by ultraviolet light with comparable frequency (Tables 3 and 9). r\‘oke that T. A to C. G transitions cannot be detected by mutation of an amino acid codon to ochre or amber: Table 10. (4) Transitions occur with particularly low frequency at T* in -C-T*- sequences (Table 3). Again, mutation to amber or ochre codons (Table 10) cannot give information on this point. (5) The virtual absence of transitions among mutations induced by 313 nm light in the presence of acetophenone (Table 7) shows that transitions are not induced TABIS

10

The sequence speci$city of mutations to amber and ochre codons induced by uttraviolet light in the E. coli lac1 gene Ochre codons

Amber codons No. of mutations

No. of mutations

NO.

Sequrncr§ Transitions 5’-PwCyt*-PurmT.(‘*. .(‘-(W Transvsrsion~ 5’.Pur-Pyr*-PurPur-T*-C.T.(‘*. -T-7’*. Totals

of sites11

NO.

C&MT uvr +

T&Gf WE-+

uvr

-

of sites11

C&MT ‘UVT+

T&G3 uvr+ uvr -

2 5 7

3 143 73

0 84 35

3 58 87

9 3 0

27 153

16 61

22 47

5 3 9 5

c2; 39 48

1 3 25 13

7 3 28 7

5 3 9 9

9 13 43 41

7 1 19 1

7 4 18 16

161

193

286

105

114

334

t Data from Coulondre & Miller (1977). $ Data from Todd & Glickman (1982). Their data for codons 06 and 07 have been omitted, since t.hese sites have not been definitely assigned. 0 Pyr* indicates the pyrimidine of the mutated base-pair. 11The numbers of sites are those for which data are reported by Coulondre & Miller (1977). Todd & Glickman do not give data for the low frequency sites, amber X9 and ochre Y3, Y4, Y6 and Y8, so the numbrrs of sites are lower, for their results.

2x8

I<.

I).

\VOOI).

‘I’.

I!

Sliol’tCK

ANI)

F

H 1.‘1‘(‘H ISSON

rfic~ientlg by ‘I’ = 1’ cyclobutyl pyrimidine dimers. present at a level c~omparahle to that induced hy 254 nm light (Tabk 8). Our results strongly suggest that t)ransitions occur when a l)Nr\ j)olytnerascB repliaat’es a template that contains Pyr(B--4)Pyo phot,oproducts (Fig. 1). Brash & HaselGne (1982) have suggest)ed that this product, is mutagenic. The produc.1 forms in double-stranded f>NA only at adjacent pyrimidines, satisfying conclusion (1) above. The product> is not formed hy 313 nm irradiation in t hfs presence of acetophrnone (Rahn & Patrick. 1976). in accordance with concalusion (5). The group on (1-4 of the 3’.pyrimidine of the product cannot participat’e in LVatson-Crick pairing (Fig. I), whereas the same group on the Spyrimidine can still do so; this is consistent with conclusion (2). Th e Iow yield of the (6-4) product at 5-CT- seyurnces in double-stranded DNA (Franklin rt al.. 1982) presumably accounts for conclusion (4). (b) Oth,er typs

qf mutations

Conclusions concerning mechanisms for mutations other than transitions are limited by the smaller amount of data. There are. however, the following points of interest. (i) Transversions The strongest evidence that these are correlat’ed with Pyr-Pyr sequences (i.e. likely photoproducts) is the low number at Pur-Pyr*-Pur sequences in the Zacl gene shown in Table 10 for three sets of data. The correlation with Pyr-Pyr may be real, but any conclusion that transversions occur preferent’ially at these sequences is less secure than the analogous conclusion for transitions. First, in ail three sets of ZacI data (Table lo), half or more of the Pyr-Pyr sites have only O-3 transversions (in the same range as numbers of transversions at Pur-Pyr*-Pur sites); in the same data sets, essentially all Pyr-Pyr* sites have many more transitions than any Pur-Pyr*-Pur site. Second. transversions in the lambda cI gene (Table 3) and the lac promoter (Table 9) are consistent, with preferential occurrence at Pyr-Pyr sequences, but are too few in number to draw statistically significant conclusions; in contrast, significant correlations between transitions and -Pyr-Pyr- sequences are found in the data for all three genes. Transversions are formed with high specificity by 313 nm irradiation in t#he presence of acetophenone (18 out of 22 mutat,ions. Table 7) but the mechanism is unknown. The major photoproduct identified to date is T = T, with a small 1979: Lin & Howard-Flanders, of T = C’ (Rahn, 1976). percentage Experimentally, there were 16 C .G to Pur . Pyr and two T. A to Pur. Pyr transversions, the converse of that expected if transversions occurred at the sites of cyclobutyl dimers. One possibilit’y for the causative lesion is suggested by the state of observation (Charlier & Helene, 1972) that the excited triplet acetophenone can abstract a hydrogen atom from guanine. The resulting guanyl free radical might readily add a bulky side group similar to those that have previously been implicated in the formation of transversions (e.g. see Eisenstadt et al.. 1982).

TARGETED

MUTAOENESIS

BY t’LTRAVIOLET

LIGHT

389

(ii) Frameshift mutations We have shown (Wood & Hutchinson. 1984) that unirradiated lambda phage assayed in irradiated host cells (i.e. non-targeted mutagenesis) yields mostly frameshift mutations. Thus, it is reasonable to ask if the frameshift mutations in targeted mutagenesis are mainly t’hose from the accompanying non-targeted mutagenesis. Son-targeted mutant frequencies for the phagr listed in Table 1 should be approximately the same as mutant frequencies for non-irradiated phage in the irradiated host cells, about 9 x lo-’ mutants/p.f.u. (footnote, Table 1). The rate x (0.74 of targeted mutation for frameshifts is (460 x lo-’ clear mutants/p.f.u.) c1 mutation/clear mut’ant) x (7 frameshift mutations/62 mutations) = 38 x 10-5/p.f.u. From the statistical uncertainty in the small number (7) of frameshift mutations, the lower 95% confidence limit is 19 x 10m5 frameshift mutations per p.f.u., significantly greater than the total rat)e for non-targeted mut.agenesis. Also. the sites and the numbers of base insertions and deletions are given in Table 5 for targeted (t,op line) and non-targeted (third line) mutagenesis: statistical tests show a low (less than 576,) probability that the two distributions could be random samplings of the same population of frameshift mutations. Thus. at least some of the frameshift mutations in Table 1 are caused by photoproducts in the phage DEA. (iii) Double mutation events Table 6 gives detailed sequence information on three mutants that were base changes at two adjacent base-pairs and three that were a frameshift accompanied by a nearby base change. LeClerc & Istock (1982) found that, of 40 mutations induced by ultraviolet light, two were adjacent base changes and one a frameshift plus a base change. In addition, mutational events with two base changes have been detected in several studies, but using procedures that would not detect double mutations involving a frameshift. With ultraviolet light, there are many cases of t,wo adjacent base changes (Coulondre & Miller, 1977; Dunst et aE., 1982), and non-adjacent but nearby pairs of base changes (Coleman et al., 1980). Single cases of two adjacent base changes have been reported: after treatment with 4-nitroquinoline oxide (Coulondre & Miller, 1977), with heat to form an apurinic site (Schaaper et al.. 1983), with X-rays (Brandenburger et al., 1981); and for nonirradiated phage in irradiated host cells (Brandenburger et al., 1981). In all of these cases, the cells had been treated to induce functions repressed by 1exA (see Introduct’ion). It was originally suggested (Witkin, 1976) that adjacent base changes could occur at t’he two bases involved in a pyrimidine-pyrimidine photoproduct. While some of the double events induced by ultraviolet light’ could be ascribed to this mechanism, some cannot. Some double mutations occur at Pm-Pyr sequences. and some at non-adjacent bases (Table 6. LeClerr & Istock. 1982; Dunst et al.: 1982). Other possible mechanisms are: (1) lesions that may lead to a temporary loss of fidelity in semiconservative or repair replication (Hopfield, 1980); (2) two lesions that happen, by chance, to occur close together may have a very much

290

II.

I). \VOOl).

‘I’

I:. SKOI’lCK

;\SIJ

1: HI.‘I’(‘HlSSOS

higher probability of bring mutagenic~ than a single isotatrti lesion: (X) sonu’ rnut,agSenic agents may tend to induce lesions in c~lustrrs (Brunk. 1973: Reynolds. 1983). These double mut,ations are highly unlikely to revert either spontjaneously or 1)) t,reatment with other mutagens. Thus. they shouttl resemble deletions in having an unmeasurable low reversion rate and the property of spanning more than one

site on a genet~ic map. We acknowledge with gratitude the expert and indispensable help of .Judith St’ein in this research. We also acknowledge with many thanks the gift of T4 endonuclease from T. Bonura and E. Friedberg (Stanford University), the sequences of a number of r1 mutants from R. T. Sauer (M.I.T.), much useful discussion with Kenneth Tindall (Yale University) and Jeffrey Miller (University of California at Los Angeles), and the patience of Mrs Estelle MacKinnon for typing the manuscript so many times. This research was supported by grant GM28297, National Institutes of Health, Department of Health and Human Services, and by contract EVO 3571 with the Office of Health and Environmental Research, Department of Energy.

REFERENCES Bagg, A., Kenyon, C. .J. t Walker. G. C. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 57495753. Belfort, M., Noff, I>. & Oppenheim. A. B. (1975). Virology, 63, 147-159. Brandenburger, A.. Godson, G. N., Radman, M., Glickman. B. W., van Sluis, C. A. &, Doubleday, 0. I’. (1981). Nature (London), 294, 180-182. Brash, D. E. & Haseltine, W. H. (1982). Nature (London), 298, 189-192. Brunk, C. F. (1973). Nature New Biol. 241, 74-76. Calvert, J. G. & Pitts, J. N. Jr (1966). In Photochemistry, chapt 7, Wiley. New York. Charlier, M. & Helene, C. (1972). Photochem. Photobiol. 15, 71-87. Cohn, W. E., Leonard, N. J. & Wang, S. Y. (1974). Photochem. Photobiol. 19, 89-94. Coleman, R. D., Dunst, R. W. & Hill, C. W. (1980). Mol. Gen. Genet. 177. 213-222. Coulondre, C. & Miller, J. H. (1977). J. Mol. Biol. 117, 577-606. Davis, R. W., Botstein, D. & Roth, J. R. (1980). In Advanced Bacterial Genetics, pp. l34137, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Devoret, R. (1965). C. R. Acad. Sci., Paris, 13, 1510-1513. Dunst, R. W., Coleman, R. D.. Harnish, B. W. & Hill. C. W. (1982). Mol. Gen. Genet. 184, 445-449. Eckardt, F., Teh, S-J. & Haynes, R. H. (1980). Genetics, 95, 63-80. Eisenstadt, E., Warren, A. J., Porter, J., Atkins, D. & Miller, J. H. (1982). Proc. Nat. Acud. AX., IJ.S.A. 79, 1945-1949. Elledge, S. J. $ Walker, G. C. (1983). J. Mol. Biol. 164, 175-192. Foster, P. L.. Eisenstadt, E. & Cairns, J. (1982). Nature (London), 299, 365-367. Franklin, W. A., Lo, K. M. & Haseltine, W. A. (1982). J. Biol. Chem. 257. 13535513543. Garces, F. & Davila, C. A. (1982). Photochem. Photobiol. 25, 9916. Hall, J. D. & Mount, 1). W. (1981). Progr. Nucl. Acid Res. Mol. Biol. 25. 53-126. Haseltine, W. A. (1983). Cell, 33, 13-17. Hecht, M. H., Nelson. H. C. M. & Sauer, R. T. (1983). PTOC. Nut. Acad. Sci., U.S.A. 80, 2676-2680. Hoplield, J. ?I. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 5248-5252. Ichikawa-Ryo, H. & Kondo, S. (1975). J. Mol. Biol. 97. 77-92. James, A. P. & Kilbey, B. J. (1977). Genetics, 87, 237-248. Kato, T. & Nakano, E. (1981). Mutat. Res. 83, 307-319. Kato, T. & Shinoura, Y. (1977). Mol. Gen. Genet. 156, 12ll131.

TARGETED

MUTAGENESIS

BY ULTRAVIOLET

LIGHT

2n1

Kenyon. C. J. & Walker, G. C. (1980). PTOC.Nat. Acad. Sci., C1’S.A. 77, 2819-2823. Krasin, F. & Hutchinson, F. (1978). Biophys. J. 24, 645-656. Lamola. A. A. & Yamane, T. (1967). Proc. Nat. Acad. Sci.. U.S.A. 58, 443-444. Lawrence, C. W. & Christensen, R. B. (1979). Mol. Gen. Genet. 177, 31-38. LeClerc. ?J. E. & [stock, N. L. (1982). Nature (London), 297, 596-598. Lin, P.-F. & Howard-Flanders, P. (1976). Mol. Gen. Genet. 146, 107-I 15. Lippkr. ,J. A., Gordon, L. K., Brash, D. E. & Haseltine, W. il. (1981). Proc. Nut. Acad. Sci., I’.S.A. 78, 3388-3392. Little. ,J. W. & Mount, D. W. (1982). Cell, 29, 11-22. Livneh. %. (1983). Proc. Nat. Acad. Sci., 7J.S.A. 80, 237-241. Maxam. A. M. & Gilbert, W. (1977). Proc. Nut. Acad. Sci., V.S.A. 74, 560-564. Miller. J. H. (1982). CeZZ,31, 5-7. Nishioka. H. & Doudney. C. 0. (1969). Mutat. ties. 8, 215-228. Nishioka, H. & Doudney, C. 0. (1970). Mutat. Res. 9. 349-358. Pabo. (‘. 0.. sauer, R. T.. Sturtevant, ,J. M. & Ptashnr. M. (1979). Proc. Nat. Acad. Sci., I’.S.A.

76, 1608-1612.

Patrick, M. H. (1977). Photochem. Photobiol. 25, 357-376. Patrick, M. H. & Rahn, R. 0. (1976). In Photochemistry and Photobiology of Nucleic Acids (Wang. S. Y., ed.), vol. 2, pp. 35-95, Academic Press, New York. Rahn. R. 0. (1979). Acta Riol. Med. Germanica, 38, 12251231. Rfahn. R,. 0. & Patrick, M. H. (1976). In Photochemistry and Photobiology of Nucleic Acids (Wang, S. Y., ed.), vol. 2, pp. 97-145, Academic Press. New York. Reynolds, R. J. (1983). J. Cell Biochem. Suppl. 7B, 181. 276, 301-302. Sauer. R. T. (1978). Nature (London), Sauer. R. T., Pabo, C. O., Meyer. B. J., Ptashne, M. & Backman, K. C. (1979). Nature (London,),

279, 396-400.

Schaaper. R. M. & Glickman, B. W. (1982). Mol. Gen. Genet. 185, 404-407. Schaaprr. R. M.. Kunkel, T. A. & Loeb, L. A. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 487491. Shetlar. M. D. (1980). Photochem. Photobiol. Rev. 5, 105-197. Skopek, T. R. & Hutchinson, F. (1982). J. Mol. Biol. 159, 19-33. Steinborn, G. (1978). Mol. Gen. Genet. 165, 87-93. Todd, P. A. & Glickman, B. W. (1982). Proc. Nat. Acad. Sri., C:.S.A. 79, 4123-4127. Weigle. .J. (1953). Proc. Nat. Acad. Sci., IJ.S.A. 39, 628-636. Witkin. E. M. (1976). Bacterial. Rev. 40, 869-907. Witkin. E. M. & Wermundsen, I. E. (1978). Cold Spring Harbor Symp. Quant. Biol. 43. 88 I -886. Wood. R. I). & Hutchinson, F. (1984). J. &foZ. Biol. 173, 293-305. Edited

by J. H. Miller