Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase

J. Mol. Biol.

(1980) 140, 101-127

Escherichia

coli Mutants

Deficient in 3-Methyladenine-DNA Glycosylase

PETER KARRANJr,

TOMAS LINDAHL

Department of Medical Biochemistry Gothenburg University, 400 33 Gothenburg, Sweden INCRID

OFSTENG,

University

GRETHE

EVENSEN

Institute of General Genetics of Oslo, Blindern, Oslo 3, Norway

AND ERLING Norwegian Division

B.

for

SEEBERG

Defense Research Establishment Toxicology, 2007 Kjeller, Norway

(Received 28 December 1979) Two Escherichiu coli K12 mutants defective in 3-methyladenine-DNA glycosylase have been isolated following mutagenesis by N-methyl-N’-nit,ro-i-nitrosoguanidine. The mutants, which are of independent origin and have been designated tug-l and lag-Z, contain greatly reduced amounts of 3-methyladenine-DNA glycosylase activity in cell-free extracts. The defect in the lag-l st.rain is observed at 43°C but not at 3O”C, and a partially purified enzyme from this strain is urlusually heat-labile, indicating that the defect in the tag-l strain is due to a mutation in the structural gene for 3-methyladenine-DNA glycosylase. We have shown that 3-methyladenine-DNA glycosylase is responsible for the rapid removal of 3-met.hyladenine from t’he DNA of E. coli cells treated with monofunctional alkylating agents. The active release of this base is greatly impaired in the mutant strains. Both tag mutant strains are abnormally sensitive to killing by monofunctional alkylating agents and are defective in the host cell reactivation of methyl methanesulphonate-treated bacteriophage X. The tag mutation does not confer an increased sensitivity to ulbraviolet. or X-irradiation, and host cell reactivation of irradiated h is normal in these strains. Further, t’here was no increase in the rate of spontaneous mutation in a tug strain. Three-factor transductional crosses with n&l and n&A have shown that the tug-2 mutation is located at 47.2 minutes on the map of the E. coli K12 chromosome. In the mapping experiments, the tug-l mutation behaved differently and appeared to be located at 43 to 46 minutes, in a closely situated but nonadjacent gene. Possible implications of the non-identity of the tug-l and tag-2 mutations are discussed. t Present

address:

Medical

Research

Council

Cell Mutation

Urlit,

University

of Sussex,

Falmer,

England. 101 0022-2836/80/170101-27

$02.00/O

0 1980 Academic

Press Inc. (London)

Ltd.

10%

1. Introduction DNA glycosylases catalyse the cleavage of base-sugar bonds in DNA. Several glycosylases that act specifically on damaged or non-conventional nucleotide residues have recently been found. Escherichia coli cells contain two enzymes that catalyst thr removal of deaminated base residues from DNA. One releases deaminated cytosine (uracil) and the other deaminated adenine (hypoxanthine) (Lindahl, 1974; Karran & Lindahl, 1978). A purine residue with an opened imidazole ring, i.e. a substituted formamidopyrimidine: is also liberated as a free base by a, separate DNA glycosylase (Chetsanga & Lindahl, 1979). In addition, the alkylation product 3-methyladeninc is excised from DNA in free form by a fourth enzyme of this type. This enzyme was first observed as one of several activities in a preparation of E. coli endonuclease 11 (Kirtikar & Goldthwait, 1974) but was subsequently found to be a distinct DNA glycosylase free from nuclease activity (Lindahl, 1976; Riazuddin & Lindahl. 1978). The 3-methyladenine-DNA glycosylase appears to recognize specifically S-methyldAMP residues in DNA since other alkylation products including 7-methylguanine, 7-methyladenine, and 06-methylguanine, and the naturally occurring met,hylated purine residue N6-methyladenine are not excised by the enzyme (Riazuddin & Lindahl, 1978). DNA glycosylases are assumed to be responsible for the initial step in an excisionrepair pathway referred to as base excision repair. However, to date, the in viva role of glycosylases has been demonstrated only for one enzyme, the uracil-DNA glycosylase. E. coli mutants deficient in this enzyme (ung) have been isolated by nonselective mass screening of cell extracts after mutagenesis and they have been very useful in defining the physiological role of the enzyme (Duncan et al., 1978). These mutants are defective in the removal of uracil from DNA that was introduced either by misincorporation of uracil in place of thymine or by deamination of cytosine in cytosine residues leads situ. The reduced ability of u?Lg mutants to excise deaminated to an increased spontaneous mutation frequency (Duncan et al.: 1978; Duncan & Weiss, 1978; Coulondre et al., 1978), apparently because transition mutations arise when cytosine is slowly converted to uracil by spontaneous hydrolysis. In addition, the ung mutation confers slightly decreased resist.ance to deaminating agents such as nitrous acid and bisulphite (da Roza et al., 1977: Hayakawa et al., 1978; Simmons & Friedberg, 1979). Here we describe the isolation and partial characterization of two E coli mutants deficient in another DNA glycosylase, the 3-methyladenine-DNA glycosylase. These have been termed tag (three-methyladenine-DNA glycosylase) mutants.

2. Materials and Methods (a) Alkylating

agents and methylated

purines

Non-radioactive methyl metharlesulphorlate and dimethyl sulphate were obtained from Merck ; X-methyl-Iv ‘-nitro-N-nitrosoguanidine was obtained from Sigma. Radioactive [methyL3H]dimethyl sulphate (1.4 Ci/ mmol) and [methyL3H]methionine (7.5 Ci/mmol) were obtained from New England Nuclear Corp. 3-Methyladenine was purchased from Fluka AG, Switzerland, and 7.methylguanine and X6-methyladenine from Sigma.

E. coZi

TAG

MUTANTS

(b) Bacterial

103

strains

The E. coli strains used are listed in Table 1. All strains were grown at 30°C in L broth 1% (w/v) NaCl, 0.104 (w/v) glucose, (1% (w/v) tryptone, 0.594 (w/v) yeast extract,, pH 7.3). Bacteria were plated on the same medium cont’aining l.So/, (w/v) agar (L-agar plates). Minimal salts medium contained (per 1): 8.2 g Na,HP0,.7H,O, 2.7 g KH,PO,, t’o 1 g (NH,)2S04, 0.1 g MgS04.7H20, 5 mg Ca(NOs)2, 0.25 mg FeSO,. 7H,O, adjusted pH 7.2. TABLE

Bacterial Strain AR1157 AB3027

NH5016

AB1157 xth metE

PK432

NH5016

tng-1

PK432.1

NH5016 argE +

tag-l

KLl6 BW9101

Hfr PO45 KL16 d (pncA-rth)

BK2012

BW9101 tag-2

BK2101

BK2012

BK2106

F-tag-Z his argE + xth + B-K2106 nnZA

KK493 KK444 El01 w3110 P3478 AB259 KL14 KL25 KL96 KL209 KL983 PK191 KLF3/JC1552 AB1886 AB1157 adaAR1 157 ada-

?lUlA nalA nrdA

strains Origin

Description thr leu proA his thi argE lac gal nrrc xyl mtl tsx str sup-37 ABl157 sth polA

BK2107

1

tag+

P. Howard-Flanders

Mutagenized derivative of AB1157 Pl transductant of AB3027 Mutagenized derivative of NH5016 KL25 x PK432, faster growing derivative of PK432 Spontaneous pncA of KL16 Mut.agenized derivative of BW9101 Revertants of BK2012 BK2012 x AB1157 Pl transductant (KK493 x BK2106)

AB 1157 uvrA

Ljungquist (1976)

et al.

Ljungquise

et al. (1976)

This work This work

Low (1972) White et nl. (1976) This work This work This work This work B. B. B. B. I>.

nrdA

W3110 poZA1 Hfr PO1 Hfr PO68 Hfr PO46 Hfr PO44 Hfr PO18 Hfr PO53 Hfr PO66 F’(P044)

Source and/or reference

thyA

M. SjGberg M. Sjiiberg Bachmann Low Rupp

Hfr kit, Low (1973) win B. Bachmann F103 his+

Low (1972) via B. Bachmann I’. Howard-Flanders P. Jeggo P. Jeggo

104

P. K,\RRAN (c) Xutagenesis

and

ET

mutant

dL selection procedure

In the initial phase of this work, E. coli mutants were sought that were deficient, either itI 3-met,hyladenine-DNA glycosylase (Riazuddin & Lindahi, 1978) or iii endonuclease IV. which acts at apurinic sites in DNA4 (Ljungquist, 197i). For this reason, E. coli zth strains were employed as parental strains, since they are deficient in the major ondonuclease activity for apurinic sites in DNA (Yajko & Weiss, 1975). (i) Isolation

of tag-l

Exponentially growing E. coli NH5016 were harvested and treated with N-methyl-N’nitro-N-nitrosoguanidine (1 mg/ml) in Tris/maleic acid buffer at pH 6.0 as described by Adelberg et al. (1965). After segregation of mutant progeny by an additional growth period, randomly selected mutagenized colonies were replica-plated on L-agar plates with and without 1.2 mM-met~hyl methanesulphonate. After 24 h growth at either 30°C or 43”C!, methyl methanesulphonate-sensitive clones were selected and re-streaked on methyl methanesulphonatr-containing plates t,o confirm sensitivit,y. Mutant’s sensitive to the alkylating agent occurred at a frequency of 6x 10F3. Altogether 65 strains sensitive to methyl methanesnlphonate at both 30°C and 43°C were isolated as well as 28 strains sensitive only at 43°C. All sensitive strains were subsequently screened for resistance to ultraviolet light. Irradiation of exponentially growing cultures of each mutant strain was carried out at 0°C in 0.05 M-sodium phosphate buffer (pH 7.4), at a dose rate of 1.3 J mm2 s-l. The total incident dose was 38 J rnm2. Survival was determined after overnight growth on L-agar at 30°C or 43°C. The survival of the parent strain E. coli NH5016 uas 2504 and that of the closely related polA strain E. coli AB3027 was 0.196. Strains which exhibited survival of > 196 were scored as ultraviolet-resistant. The overall frequency of such mutants, unable to grow in the presence of 1.2 mM-methyl methanesulphonate biit showing little or no increased sensitivity to ultraviolet irradiation, was 2 x 10 -3. (ii) Isolation

of tag-2

Exponentially growing BW9101 were mutagenized as described above for NH5016. After mutant segregation, the mutagenized stock cultures were screened for auxotrophs which, after effective mutagenization, amounted to 25 to 30% of the cell population. Mutagenized cell cultures were grown on agar plates containing minimal salts medium supplemented with 0.5:/, glucose, and colonies were picked after 2 days incubation at 30°C. The isolates were further grown in L-broth in 96.well plastic depression plates and replica-plated on L-agar by means of a 4%prong replicator to test for sensitivity to methyl methanesulphonate, X-irradiation, and nltraviolet light. The mutagenizatiori and screening tests were part of a general approacli to isolate DNA repair mutants t,hat were deficient, in early steps in t,he excisionrepair of various types of DNA damage. About 50 mutants that did not grow on plat,es containing 2.2 mlw-methyl rncthanesulphonat~r were further tested for host cell reactivatiorr of bscteriophapr X treated with 0.05 x-methyl methanethis treatment’ sulphonate for 20 min at 37°C. .4pproximatcly 22”i; of the phage survived when plated on the parelit strain BWRlOl. In 4 of the mutants tested, phage survival values were reduced. The mutant most deficient in host) cell react’ivation (2% survival of t,he alkylated phage), strain BK2012. was subjected to further analysis by measurements of enzyme activities in cell extracts. (d) Quantitative (i) Cell survival

in liquid

assays

for sensitivity

to alkylating

agent.s

cultures

Exponentially grow-ing cultures were harvested by centrifugation and suspended at approx. 10s cells/ml iii minimal salts medium prewarmed to 30°C or 43°C. Methyl methanesulphonatc was diluted immediately before use in minimal salt,s medium at 0°C and added to the bacterial susperrsiou to give a final concn of 0.02 M. Portions of the culture were removed at several different times, diluted, and plated on prewarmed L-agar plates. Colonies were counted after 24 h of incubation at either 30 or 43°C.

E. coli A similar protocol nitro-hT\--nitrosoguanidine (ii) Paper

disc

was followed at 30°C.

TAG

105

MUTAKTS

for treatment

of cultures

with

0.7 miW-hi-methyl-i!Y’-

method

The method described for sensit,ivity to methyl (iii) Host cell reactivation.

by Ljungquist et al. (1976) was used as a rapid methanesulphonate. methanesulphonute-treated

of methyl

bacteriophage

quantitative

assay

h

Exponentially growing cells were harvested and concentrated 4-fold in X suspending buffer (Miller, 1972). Then 0.2 ml cell suspension was mixed with 0.1 ml of appropriate dilutions of bacteriophage Xc1857 treated with 0.05 M-methyl methanesulphonate at 37°C for the times indicated. The mixtures were incubated for 10 min at room temperature to allow phage adsorption and plated on L-agar in a 0.6% top agar layer. The alkylation treatment of the phage ( lo8 phage/ml in h suspending buffer) was stopped by dilution of the phage suspension by at, least loo-fold. (e) Sendivity

(i) Ultraviolet

to other

agents

irradiation

Bacteria were suspended at approx. lo8 cells/ml in minimal salts medium. Cells were irradiated with 254 nm light from a low pressure mercury lamp at an incident dose rate of 1.3 Jm-2s-1. (ii) X-irradiation Growing bacteria were harvested and suspended in 0.05 M-sodium phosphate buffer (pH 7.4). X-irradiation was carried out in air at 0°C at a dose rate of 1 krad min-‘. Portions of the irradiated bacterial suspension were removed at various time intervals, diluted in ice-cold 0.05 M-phosphate buffer (pH 7.4), and plated immediately. The surviving fraction was determined after 24 h of incubation. (iii) Nitrous

acid treatment

Exponentially growing cells were suspended in 4.75 ml minimal salts medium, and 0.25 ml of a solut,ion of 0.2 M-NaN02 in 2 M-sodium acetate buffer (pH 4.3) was added. Control cultures received acetate buffer alone. Samples were removed and plated at different time intervals. Survival was determined after 24 h of growth. (iv) Host cell reactivation

of ultraviolet

or X-irradiated

bacteriophage

X

Phages were plated with exponentially growing cells as described above for host cell reactivation of alkylated A. For ultraviolet irradiation the phages were suspended in h buffer and exposed to 254 nm light at a dose rate of 1 J me2 s-l. For X-irradiation. phages were suspended in L-broth and exposed to X-rays at 0°C at a dose rate of 2.0 krad min-I. (f) Isolation

of revertants

Revertants of strain BK2012 were obtained by plating lo9 stationary phase cells on plates containing 2.6 mm-methyl methanesulphonate followed by incubation at 37°C overnight. Revertants, occurring with a frequency of about 1O-7, gave rise to large colonies that were clearly different from the small colonies obtained from surviving nonrevertant bacteria. (g) Enzyme (i) Preparation

assays

of cell extracts

Bacterial cultures of 250 ml were harvested in the logarithmic growth phase and washed with cold 0.05 M-sodium phosphate buffer (pH 7.4). The pelleted cells were stored frozen

106

t’

KA It R .! Y / E 7’ :I F,

at, - 70°C. Thawed cells wore disrupted by grindin, 17with sea sand and extracted wit,11 3 vol. 0.05 nl-Tris~HCl (pH 7.3), 1 m~-~IYl?~4, 0.1 111M-dithlot)lI.(‘itOl. G,td ztntl cell debris \Y<‘IY’ removed by 2 successive ct,llt,rifu~atiorls at, 10,000 g and 23,000 g for 10 rnitl c~trll. ‘1’11~ superrratant (crllde ext,ract, fractiorl I) was made 0.80,, iri stjrcptomvcirl s~llpllatt~ tjy th(% dropwiso addit,ion of an equal vol. of a 1.6” f0 solntion it1 tile same t,ufft:r w+th cc,rrst;tnt stirring at 0°C’. After 20 lnin of additional stirrittp, t,krc precipitdtn writs rcmov(:d t)y cerltrifugatiolr (15 Inil), 23,000 g). The silprrriatarlt, solution was fract ioliatntl by t,lrt, addition of solid ammotii1im sulphate and the prot.eiti precipitating bctwc‘c‘lr 40!/; and 7OU, saturation (I.6 to 2.8 M) was dissolved in a small vol. of 0.05 b[-Tris.HCl (pH 7.5). 0.3 MNaCl, 1 mw-EDTA, 0.1 Inns-dithiot,hreitol, and dialysed for 16 h at 4°C against the same bufFer. This dialysed ammonium sulphatc fraction (fractiotl II) was 11set1to scrocn rnutarlt sxt)racts for 3.lnctl-lylstlcrlirle-DNA plycosylascb and r~~tlo~~~~clsase 1V activities. (ii) Assay

procedures

:~-Methyladenine-DNA plycosylase was assayed t+ssentially as described by Riazgiddirl dz Lindahl (1978). The standard react.ion mixture ( 100 ~1) contained 0.07 M-HEPES .KOH (pH 7.8), 1 rnnf-EDTA, I rnl\~-tlitlliotllreitol, 5’+,, glycerol, IO p-1~:1,rrethyl-3H]diInctllyl slill)llate-trrat,rd DNA (4000 cts/min) and enzyme (5 to 20 pg protein). Llcubatiotr was carried out for 30 min at 3O”C, followed by chilling t,o 0°C and addition of 10 ,LI heat denat,ured 0.20, calf thymus DNA, 10 ~1 2 hr.NaCl, and 300 ~1 cold ethanol. After mixing. samples were k(xpt at, -220°C for 20 min prior to cLEDTA, O-l rn~r-dithiothn:itol, 25 pg ho~in(: serum albumin, 7.5 pg I’M2 [“H]DNA (6000 ct,s/min) and 0.5 t,o IO pf: E. coli extract). .Yftcr incuh&iotl at 30°C for 30 min. t’lrc rea&on was stoppod by the addition of 409 ~1 0.3 Rr-potassiunl phosphate (pH 12.1). Aftclr reduction of the pH by addition of 200 ~1 2 &I-Tris.HCl (pH i.5). single-stranded DNA was trapped The filters were washed with 0.9 >r-NaC’l, by filtration through nitrocellulose filters. 0.09 1%.sodium citrate (pH 7.0), tlripd, and counted ir, 5 ml of tolur:nr-t)iLsf?d scintillation fluid. In screening cell extracts for the possible presence of a telnparatum-sensitiv,~ endonuclease IV, the same assay was carried-out in parallel at both 30°C and 43“C. The endon~~lrasr functior\ of E. coli exonuclease II I (Weiss. 1976) ~vas tneas~~r~d by tile same assay used for c~ndormclrase IV, tlxcept t~hat the rt?act.ion mixt,,lrr did not, contain EDTA or NaCl. (h) Partial

purijication

of a heat-labile

3-methyladenine-DNA

glycosylase

A heat-labile 3-methyladenine-DNA glycosylase activity was partially purified from X. coli PK432. Cells (3 g), harvested from cultures in exponential growth at 3O”C, were disrupted by u!trasonic treatment in 15 ml extraction buffer. After centrifugation, the extract was treated with streptomycin and fractionated with ammonium sulphate as described above. The material that precipitated at 70% saturation with ammonium sulphate was dissolved ill 1 ml 1 M-NaCl, 0.01 wHEPES.KOH (pH 7.4), 1 mM-EDTA, 1 mwdithiothreitol, 5% glycerol, and applied to a column of Sephadex G75 (1 cm x 100 cm) equilibrated with the same buffer. Fractions containing 3.methyladenine-DNA glycosylaso

E. coli activity further resulted

TAG

were pooled (fraction III). This purification by phosphocellulose in loss of activity. (i) Chain

(i) Alkylation

enzyme fraction chromatography

cleavage of alkylated

107

MUTASTS

DXA

was unstable and attempts at (Riazuddin & Lindahl, 1978)

by crude cell extracts

of DNA

Covalently closed 3H-labelled ColEl DNA (16,000 cts/min per pg) (Seeberg, 1978) was treat,ed with 10 mi\l-methyl methanesulphonate in 10 mM-Tris.HCl (pH 8.0), 1 mM-EDTA for 15 min at 37°C. The alkylating agent was removed by gel chromatography on Ultrogel ACA 44 (LKB) equilibrated with the same buffer. (ii)

Preparation

qf extracts

Cell extracts were prepared from combination of sucrose plasmolysis et al. (1976). (iii)

Reaction

conditions

and strand

40 ml cultures and lysozyme

of exponentially growing cells by a treatment’ as described by Seeberg

break measurements

The reaction mixtures contained 100 mM-KCl, 1 rnN-dithiothreitol, 5 mM-MgSO,, 50 mMmorpholino-propane sulphonic acid (pH 7.5), 0.05 pg DNA and 20 ~1 ext,ract (-40 pg protein) in a total vol. of 140 ~1. After 20 min at 37”C, t’he reaction was stopped by adding an alkaline buffer (2.5 ml 1 M-NaCl, 20 mM-EDTA, 50 miv-sodium phosphate, adjusted to pH 11.9 with NaOH) which selectively denatures DNA that contains strand breaks. The fraction of cleaved DNA was measured after neutralization and filtration t’hrough nitrocellulose filters (Center et al., 1970). (,j) Excision

of methylated

bases from

DSA

alkylated

in viva

Both tag mutant strains were tested for the ability to excise 3-methyladenine from their DNA in vivo. E. coli BW9101 (tag+) and BK2012 (tag-&) were assayed at 30°C while E. coli PK432 (tag-Z) was tested at its restrictive temperature of 43°C. Overnight cultures were diluted into L-broth and grown with aeration to approx. 108 cells/ml. Cells from a 1 1 culture were collected by centrifugation and suspended in 20 ml prewarmed minimal salts medium. [“Hldimethyl sulphate was added to each cultural to a final concn of 0.56 rnM (112 &i/ml). After 10 min of alkylation, two 3-ml portions were rrmoved for zero-po;nt analysis of alkylation products. Further alkylation was prevented by rapid chilling on crushed ice. The remaining cells from each culture were dilut,ed 80.fold in prewarmed minimal salts medium. Portions of each culture were harvested at different times after treat,ment. The surviving fraction of bacteria was determined at each time-point after alkylation by plating appropriate dilutions of cell suspension on L-agar. Crll lysis and DNA extraction were carried ollt immediately after harvesting the cells. DNA was extracted frorn the crlls by a modified Marmur (1961) procedure designed to eliminate prolonged incubations at low pH and at temperatures ahove 0°C in order to minimize loss of 3.methyladenine from DNA by spontaneous depurination. Cells wer? lysed by treatment with lysozymc (1 mg/ml, 20 min at OOC) followed by the addition of 1 ml 5% Sarkosyl (Ciba) in 0.5 M-Tris.HCl (pH 9.0) and mcubation for a further 15 min at 0°C. A viscous lysate was obtained to which 1.5 ml 5 M-NaClo, and 10 ml chloroform/ isoamyl alcohol (24 : 1, v/v) were added. After vigorous mixing, the phases were separated by centrifugation (24,000 g, 10 min) and the DNA was precipitated from the aqueous phase by the addition of 0.1 vol. 2 M-NaCl and 2 vol. ethanol (pre-chilled to -20°C). The DNA was recovered by spooling on a glass rod and dissolved in 2 ml cold 0.01 Ivr-Tris . HCl (pH 8.0), 0.001 nr-EDTA. The resulting viscous solution was digested with 200 llg pancreat,ic RNase/ml (Worthington, preheated to remove traces of DNase) for 10 min at 37°C. After cooling to 2O”C, 200 ~1 3 M-potassium acetate, 1 mM-EDTA were added and tile DNA was prcacipitated by the slow addit,ion of 0.5 to 0.6 vol. cold isopropanol. The

1’. KARKAN

108

ET

.-1 I,.

precipitate was recovered by spooling on a glass rod, washed extensively in 707; ethanol, dissolved in 2 ml 0.3 M-potassium acetate, 0.01 M-Tris.HCl (pH 8.0), 1 mu-EDTA, and the isopropanol precipitation was repoated once. Thr DNA precipitate was again wa.shed extensively in 70% ethanol, and dissolved in 600 ~1 1 mM-phosphate (pH 7.1). Purified DNA was hydrolysed at neutral pH (pH 7. I, lOO”C, 30 min) to release alkylated purines and the total hydrolysates were applied to Whatman 3MM chromatography paper with authentic 7-methylguanine and 3-methyladenine as markers. Following chromatography for 24 h in isopropanol/conc. aqueous NH,/water (7 : 1 : 2, by vol.), the dried papers were cut transversely int.o 1 cm strips and the radioactive material cochromatographing with the marker compounds was determined. (k) In viva

radioactive

labelling

of ,methylated

bases in DNA

Overnight cultures of E. coli BK2012 (tag-2) and E. coli BW9101 (tag+) (5 ml of each) in minimal salts medium supplemented with 0.5% glucose and O.Z”,e yeast extract were diluted into 250 ml of the same medium. After 2 h growth at 3O”C, 5 mCi [methyL3H]methionine was added to each culture together wit11 2-5 ml 1 mrvr-thymidine and 2-5 ml 1 mM-xanthine. The cultures were shaken for 2 11, during which time growth was normal as monnored by Ae4e nm measurements. The bacteria were then harvested, washed with 50 ml ice-cold 0.05 M-potassium phosphate buffer (pH 7.4), and suspended in 5 ml ice-cold 0.02 M-NaCl, 0.01 M-EDTA, 0.01 M-Tris’HCl (pH 8.0). DNA was extracted by the rapid method outlined above and hydrolysed at pH 7.1 (100°C for 30 min) to release selectively any 3.methyladenine present. The remaining DNA was acid-precipitated and subsequently hydrolysed in 0.1 M-HCl at. iO”C for 30 min to release remaining purme residues, including X6-methyladenine. The hydrolysates of DNA from the 2 cultures were analysed by paper chromatography. The chromatograms were developed by descending chromatography for 24 h in isopropanol/conc. aqueous NH,/water (7: 1: 2, by vol.). The areas of absorption corresponding to X6-methyladenine and 3.methyladenine were cut out. eluted with 2 ml water, concentrated by evaporation, and rechromatographed in the same system. The dried chromat,ograms were then cut transversely int,o 1 cm st>rips from which the radioactive material was elut,ed and quantitated. ( 1) Spontaneous

mutation

frequency

Frequencies of spontaneous mutations to rifampicin and ampicillin resistance were determined by a modified fluctuation test. as described by Duncan et al. (1978). Cultures were grown to stationary phase from a single colony in L-broth containing 0.1% glucose. Ten different tubes of the same medium were inoculated with 100 to 200 cells from the stationary phase culture and grown without aeration at 37°C overnight. Spontaneous mutants were selected by plating 0.5 ml of each subculture in 0.8% soft agar on L-agar containing 100 pg rifampicin/ml or 10 pg ampicillin/ml. Viable cells were counted after combining 0.1 ml of each subculture and plating at appropriate dilutions on L-agar. (m) Genetic mapping

procedures

Procedures used for conjugation and Pl transduction were those described by Miller (1972). In most transduction experiments phage Pl (607H), a clear-plaque mutant which yields larger numbers of transductants than normal Pl phage (Wall & Harriman, 1974), was used. Minimal plates contained minimal salts medium supplemented with 0.2% glucose, appropriate amino acids, and 1.5% (w/v) agar. The MMSR/MMSSt phenotype of the tug+/tagbacteria was scored by replica plating drops of stationary phase cultures, grown in plastic depression plates, on L-agar containing 3 mM-methyl methanesulphonate. Both wild-type and zth strains gave a confluent area of bacterial growth on such plates, while all tag mutants used were unable to grow. 7 Abbreviation

used : MMSR, MMSS, methyl

methanesulphonate

(resistant,

or sensitive)

E. coli

TAG MUTAKTS

109

using a high multiplicity of infection (0.1 to 1.0) Transduction to r&AR was performed to maximize the yield of transductants relative to the number of spontaneous r&AR mutants (Duncan et al., 1978). Phage readsorption was prevented by addition of citrate and the NalAR phenotype (which is recessive to NalAs) was expressed by incubatmg t-he transduced bacteria for 3 h in broth before plating on L-agar containing 40 pg nalidixic acid/ml. nrdA+ transductants were selected on appropriate minimal plates by incubating at 42°C. The nrcZA-- strain employed (ElOl), conditionally lethal at 42°C but somewhat leaky, gave a large number of colonies when plated at the cell densit,ies necessary to obtain transdnctants. Colonies which were nrdA- were eliminated after retesting for growth at,

42°C.

3. Results (a) Isolation

of mutant strains

Since 3-methyladenine is a common DNA lesion introduced by treatment with $ Brookes, 1963), it was expected that an E. coli strain alkylating agents (Lawley deficient in an enzyme specifically acting on the lesion might be sensitive to methyl methanesulphonate. Therefore, mutagenized E. coli clones were tested for sensitivity to methyl methanesulphonate on agar plates that contained low concentrations of the alkylating agent, and clones were selected which showed reduced survival compared to the parental strain. The same clones were also screened for sensitivity to ultraviolet light and those were excluded which showed marked sensitivity. The latter procedure was employed to eliminate previously well-characterised mutants, such as recA, recB, lex and poEA, which are defective in functions generally involved in repair of DNA damage and are sensitive to most DNA damaging agents. Since 3-methyladenine has never been detected in ultraviolet-irradiated DNA, mutants deficient in 3-methyladenine-DNA glycosylase were expected to be radiation-resistant. The isolates sensitive to methyl methanesulphonate and resistant to radiation were further tested either for DNA glycosylase activity by enzyme assays of partially purified extracts (derivatives of NH5016) or for host cell reactivation of bacteriophage h that had been treated with methyl methanesulphonate (derivatives of BW9101). Partially purified extracts from 36 different clones derived from strain NH5016 were screened for 3-methyladenine-DNA glycosylase and endonuclease IV activities. In 35 of the 36 strains, the 3-methyladenine-DNA glycosylase activity was found to be 108*25% of the level in the parent strain. However, one strain (PK432, tag-l) was isolated which had markedly reduced levels of this enzyme when assayed at 43°C. No mutants defective in endonuclease IV were detected. The mean value for the activity of that enzyme in the different extracts was 106f 30% of the level in extracts of the parent strain. In a different approach, mutants sensitive to methyl methanesulphonate were screened for their capacity to reactivate alkylated bacteriophage A. By analogy with the uvr mutants from E. co&, which are deficient in host cell reactivation of ultravioletirradiated phage (Howard-Flanders et al., 1966), tug mutants were expected to be Her- for methyl methanesulphonate-treated phage. One such mutant (BK2012, tag-Z) was isolated as a strain highly deficient in reactivation of alkylated phage A. Cell extracts of this mutant were subsequently found to have a greatly decreased level of 3-methyladenine-DNA glycosylase activity.

30 (0)

600 30 Exposure time hni

60

(b)

FIG. 1. Sensitivity of E. coli lag mutants to methyl methanesulphonate as measured by survival in liquid cultures. Exponentially growing cultures at 30 and 43°C of ~ng mlltant,s and their parent strains were harvested and resuspended in minimal saks medium of the same temperature. Methyl methanesulphonate was diluted immediately prior to use in ice-cold mimmal salts medium and added to the bacterial suspension to give a final concn of 0.02 M. Portions were removed at the t,imes indicated, plated on L-agar, and incubated at 30 or 43°C. (a) tag-l mutant. Triangles, E. coli PK432 (zth fag-l); circles, E. coli SH5016 (zth tczg+); open symbols, survival at 30°C; closed symbols, survival at 43°C. (b) tag-2 mutant. Triangles, E. coli BK2012 (zth tug-Z); circles, E. coli BW9101 (xth tag’); open symbols, survival at 30°C; closed symbols, survival at 43°C.

(b) Properties

of E. coli tag mutants

The mutant strain E. coli PK432, derived from strain NH5016, showed resistance similar to the parent strain on exposure to 0.02 M-Illethyl methanesulphmte at 30°C but was markedly more sensitive to the alkylating agent at 43°C (Fig. l(a)). In agreement with these observations, cell-free extracts from E. coli PK432: prepared from bacteria grown at 3O”C, contained 60 to 90% of the level of 3-methyladenineDNA glycosylase activity found in extracts of the parent, strain. However. following growth at 43”C, the level of this enzyme activity in PK432 extracts was only about 20% of the amount in extracts of the parental strain (Table 2). The mutant strain E. coli BK2012, derived from strain BW9101, was deficient in host cell reactivation of methyl methanesulphonate-treated phage X, and was isolated by this assay. Subsequent experiments showed that’ E. coli BK2012 was markedly sensitive to methyl methanesulphonate at either 30 or 43°C (Fig. l(b)). At 43°C. strains PK432 and BK2012 showed similar sensitivity to the alkylating agent. After growth at either 30 or 43”C, extracts of strain BK2012 contained only about 5% of the level of 3-methyladenine-DNA glycosylase activity present in the parental strain (Table 2). E. coli BK2012 grew at a normal rate at 30 to 43°C. The residual 3-methyladenine-DKA glycosylase activity in extracts of strain BK2012 was purified about 20-fold by ammonium sulphate fractionation and gel chromatography; it had biochemical properties different from the major enzyme

E.

coli

TAB

111

MUTANTS

TABLE 2 dmounts

of d-methyludenine-DNA glycosylase activity in ammonium fractionated cell extracts of differed E. coli strains Enzyme activity? (p unitsjmg protein)

Strain

NH5016

PK432

(zth),

sulphate-

30°C 43°C

(zth tug- I), 30°C 43°C

BW9101

(A)

BK2012

(rth,

BK2106

(zth+,

BK2012

6 tag + revertants

6.9 5.8 5.2 1.0 6.4

tag-2) fag-Z)

0.3 0.4 5.4-7.5

Growth of bacteria and assays were performed at 37°C unless otherwise stated. (Similar results were obtained with crude cell extracts. but the lower levels of activity made the determinations less quantitative.) j’ One unit of 3-methyladenine-DSA glycosylase activity catalyses the release of 1 pmol free 3-methyladeninelmin from alkylated DNA under the standard assay conditions.

activity in the parental strain. Thus, more than 90% of this activity was recovered after heating at 45°C for 15 to 30 minutes, while the major enzyme activity of wildtype bacteria was 80% heat’-inactivated after 15 minutes at 45°C. Further, remaining activity in BK2012 was resistant to product inhibition by 5 mM-3-methyladenine (less than 10% inhibition), while the major activity in tag+ strains was productinhibited under these conditions (Riazuddin & Lindahl, 1978). The alkylated base released by the heat-stable activity was analysed by chromatography and confirmed to be 3-methyladenine. A similar heat-stable 3-methyladenine-DNA glycosylase activit!y, resistant to product inhibition, was found to be present in similar amounts in the parent strain, as well as in E. coli B extracts, and accounted for 5 to loo/, of the total 3-methyladenine-DNA glycosylase activity observed under the assay conditions used. Thus, it would appear that wild-type E. coli contains two different 3-methyladenine-DNA glycosylase activities, a major form which is heat-labile and sensitive to product inhibition, and a minor, more heat-stable form. E. coli BK2012 lacks detectable amounts of the former activity but has the same level of the latter activity as other strains. The data shown in Figure 1 were obtained with E. coli tag mutants having an xth background. Since the xth mutation by itself confers slight sensitivity to methyl methanesulphonate (Ljungquist et al., 1976), the tag-2 lesion of strain BK2012 was moved into an xth+ strain by conjugation (see section (k), below). This strain, E. coli BK2106, had a wild-type level of xth+ gene product, assayed as the endonuclease function of exonuclease III (Yajko & Weiss, 1975). However, strain BK2106 differed

112

t 0

20 Exposure

40 time

60 (mln)

FIG. 2. Sensitivity to methyl methanesulphonate of an E. co& tag mutant having an zth+ background. E. coli BK2106 (zth+ tag-Z), BK2012 (zth tag-Z) and BW9101 (zth tag+) were harvested from exponentially growing cultures, suspended in minimal salts medium at 30°C and treated with 0.02 M-methyl methanesulphonate as described in the legend to Fig. 1. E.coZiBK2012(~);E.coZiBW9101(O);E.coZiBK2106(~).

only slightly from strain BK2012 in its sensitivity to 0.02 M-methyl methanesulphonate in liquid culture (Fig. 2). Further, extracts of strain BK2106 had the strongly reduced level of 3-methyladenine-DNA glycosylase activity characteristic of strain BK2012 (Table 2). These data show that a tug mutation is similarly expressed in either an stF,+ or an &:th strain. Moreover, the results serve to confirm the separate nature of the E. coli 3-methyladenine-DNA glycosylase (Lindahl, 1976) and the major endonuclease for apurinic sites (Verly et al., 1973; Weiss, 1976). An early report of the presence of both kinds of activities in a single enzyme, endonuclease II (Kirtikar & Goldthwait, 1974), has not been confirmed. (c) Sensitivity

of tag mutants to different alkylating radiation

agents and resistance to

Because 3-methyladenine occurs in DNA after treatment with a variety of methylating agents, tug mutants might be sensitive to other alkylating agents such as N-methyl-N’-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea that introduce a broader variety of lesions in DNA than methyl methanesulphonate (Lawley & Thatcher, 1970). The sensitivity of a tag mutant to a representative agent of this type was studied. The data in Figure 3 show that the deficiency in the tag-2 mutant confers markedly increased sensitivity to N-methyl-N’-nitro-N-nitrosoguanidine. Radiation treatment does not seem to introduce 3-methyladenine into DNA and was therefore used in the scheme for isolation of tug mutants. In quantitative studies with the isolated tag-l and tag-2 mutants and their parent strains, no difference in sensitivity to ultraviolet light was detected. Both strain PK432 (tag-l) and its parents strain NH5016 (tug+), grown at 43”C, showed 37% survival after exposure to 38 J m -2. A related poZA strain, included as a control, was fourfold more sensitive to ultraviolet

E. coli

TAG

MUTANTS

113

0Exposure

time (mm)

FIG. 3. Sensitivity of E. coli BK2012 (zth tag-Z) and its parent strain BW9101 (zth tag+) to N-methyl-N’-nitro-N-nitrosoguanidine. Cells were harvested from exponentially growing cultures W&S and resuspended in prewarmed minimal salts medium. N-Methyl-iV’-nitro-N-nitrosogusnidine dissolved in 0.06 M-sodium acetate (pH 5.0) and added to the bacterial suspension to give a final concentration of the alkylating agent of 0.7 mM. After incubation at 30°C for the times indicated, portions of the bacterial suspension were removed and appropriate dilutions were plated on Lager. E. coli BK2012 (A); E. co.%BW9101 (0).

radiation under the same conditions. Similar results were obtained with strains BK2012 (tag-Z) and BW9101 (tug+). In addition, it was found that tug mutants were not detectably more sensitive to X-irradiation than their parent strains; both tag+ and tug strains showed 37% survival after exposure to 12 krad under air (data not shown). The latter observation argues against a role of the 3-methyladenine-DNA glycosylase in the removal of some major type of DNA base lesion introduced by ionizing radiation. Treatment of isolated DNA or living cells with nitrous acid causes the deamination of DNA bases, and DNA glycosylases different from the enzyme that releases 3-methyladenine have been found which specifically remove the deaminated forms of cytosine or adenine from DNA (Lindahl, 1974; Karran & Lindahl, 1978). Within experimental error, the tag-l and tag-2 mutants exhibited the same level of resistance to killing by nitrous acid treatment as their parent strains (data not shown). (d) Host cell reactivation in tag mutants of bacteriophage X damaged by exposure to methyl methanesulphonute, ultraviolet light, or X-rays (Her) of phage treated The ability of a tag mutant to perform host cell reactivation with DNA-damaging agents outside the cell was investigated. The response of a polA strain was included for comparison. DNA polymerase I is implicated in repair of damage caused by all the agents used. The tag-2 mutant shows an Her- phenotype for methyl methanesulphonate-treated h phage irrespective of the presence or absence of an xth mutation (Fig. 4). The p02A mutant is also Her- for alkylated phage, but less so than the tag mutants. In contrast, both ultraviolet and X-irradiated phage

114

\ ‘\ -7

‘.x‘\

‘\

“\\ \\ \X\

\ IO

0

IO

20

Exposure time (mm) FIG. 4. Host cell reactivation of alkylated bacteriophage Xc1857 was exposed to 0.05 M-methyl methanesulphonate subsequently plated on E. coli strains: BK2012 (tag-2 rth) (A), P3478 (poL4I) (x), AB1157 (A).

Bacteriophage A in E. co& tag mutants. at 37T for the times indicated and ( l ), BW9101 (zth) (O), BK2106 (tag-Z)

survive normally on tug-2 bacteria, while the poZA mutant is Her- for irradiated phages as well. These responses are in good agreement with the results obtained when cells are exposed to the same agents. (e) Revertants Six independent revertants of strain BK2012 were isolated and shown to be competent to perform host cell reactivation of methyl methanesulphonate-treated phage h. These strains had also regained the resistance to treatment with 0.02 M-methyl methanesulphonate in liquid culture typical of the parent strain. Moreover, cell extracts from all six of these revertant strains were shown to contain wild-type levels of 3-methyladenine-DNA glycosylase by direct enzyme assays, and more than tenfold higher activity than extracts from strain BK2012 (Table 2). These data show that the sensitivity of strain BK2012 to alkylation is related to the deficiency in 3-methyladenine-DNA glycosylase activity. Several attempts to isolate analogous revertants from strain PK432 were unsuccessful. (f) Chain cleavage of alkylated

DNA

by extracts from tag + and tag cells

Because 3-methyladenine is the predominant lesion subjected to repair in methyl methanesulphonate-treated cells, removal of 3-methyladenine in vitro should be observed by incubating alkylated DNA with cell extracts and measuring the formation of apurinic sites and single-strand breaks in the DNA. Table 3 shows that about 0.70 strand break (or alkali-labile sites) per DNA molecule is generated by crude cell extracts from either KLl6 or BW9101 (xth) in covalently closed ColEl DNA treated with 10 mw-methyl methanesulphonate for 15 minutes at 37°C while only 0.17 strand break (or alkali-labile sites) is generated by an extract from BK2012 (tag-2, xth) under

E.

coli

TAG

115

MUTAKTS

TABLE 3 Chain cleavage of alkyluted,

Cell extract from z&rain

KL16

covalently closed circular tag-2 and tag+ cells

DNA

DNA

molecules by extract

cleaved (94)

DNA

by extracts from

Methyl methanesulphonate-specific cleavage (strand breaks/molecule)

Strand breaks/ moleculet

Untreated

2”

0.24

Alkylated

61

0.94

Untreated

18

0.19

Alkylated

59

0.89

Untreated

24

0.27

Alkylated

36

0.44

0.70 BW9101

(zth)

0.70 BK201” (tag-2 rth)

t Calculated DNA molecules.

0.17

assuming

a Poisson

distribution

of strand

breaks

in the

population

of circular

the same conditions. These data indicate that 70% of the strand breaks detected in alkylated DNA after incubation with wild-type extracts occur after the initial action of 3-methyladenine-DNA glycosylase on alkylation lesions. (g) Mutation

in the structural

gene for the enzyme

In comparison with its parent strain, the E. coli PK432 strain showed an essentially normal level of 3-methyladenine-DNA glycosylase activity when grown at 30°C and a fivefold lower level of activity after growth at 43°C. When a crude cell extract, or the approximately 20-fold purified enzyme from strain PK432, was incubated at 43°C in parallel with enzyme fractions of similar purity from the parental tag + strain, the enzyme activity present in PK432 showed increased heat lability (Fig. 5 and Table 4). These data seem in good agreement with the properties of this strain, which is much more sensitive to alkylation at 43°C than at 30°C. Cell extracts of 19 additional E. coli mutants, isolated during the search for tag mutants, that were found to be sensitive to methyl methanesulphonate at 43°C but not at 30°C and resistant to ultraviolet irradiation were also tested, but all had normal levels of 3-methyladenineDNA glycosylase, with heat stability characteristic of the wild-type strain. The heat lability observed in the enzyme in strain PK432 strongly indicates that the mutation in this strain, which confers sensitivity to alkylating agents at high temperature, is in a structural gene for 3-methyladenine-DNA glycosylase. (h) Removal of alkyluted purines from

DNA

in vivo

After exposure of E. coli to alkylating agents, 3-methyladenine residues are rapidly removed from the DNA while 7-methylguanine is not actively excised (Lawley & Orr, 1970), an enzymatic process being implicated in removal of the former lesion (Lawley & Wa,rren, 1976). Experiments with E. coli tag mutants were performed in order

116

I’.

0

KARRAN

ET

IO

5

A I,.

15

20

Time at 43°C (mln)

FIG. 5. Heat lability of 3.methyladenine.DNA PK432 and its parent strain NH5016 (tag+ ) were was purified approx. 20.fold from both strains. in 1 M-N&J, 0.01 M-HEPES.KOH (pH 7.4), Portions were removed for enzyme assays at the enzyme from E. coZi NH5016 (0).

glycosylase from E. coli PK432 (tag-I). E. coli grown at 30°C. 3.Methyladenine-DNA glycosylase These enzyme fractions were incubated at 43°C 1 mu-EDTA, 1 m&r-dithiothreitol, 5% glycerol. times indicated. Enzyme from E. coli PK432 ( A) ;

TABLE

4

Difference in heat lability of 3-methyladenine-DNA glycosylase from strain NH5016 (tag+) and PK432 (tag-l) at different stages of puri$cation

Enzyme

fraction

Crude cell extract (NH,),SOI fraction Sephadex G75 fraction Bacteria inactivation

Specific activity (p units/mg protein) NH5016 PK432 3.1 6.8 61

1.7 2.4 24

were grown at 30°C. Portions of the enzyme fractions and subsequently assayed at 30°C.

Time for 50% inactivation at 43°C (min) PK432 NH5016 28 23 16 were incubated

6 5.7 5 at 43°C for heat

to investigate if 3-methyladenine-DNA glycosylase is active in the removal of 3-methyladenine from DNA in z&o. The amounts of alkylated purines persistent in DNA after treatment of different E. coli strains with [3H]dimethyl sulphate are given in Table 5. The data for the tag+ strain BW9101 are similar to those obtained earlier for E. coli WP2 by Lawley and co-workers (Lawley & Orr, 1970; Lawley & Warren, 1976). In the first measurements made after alkylation, the ratio of 3-methyladenine to 7-methylguanine was three to four times lower in the tag+ strain than that observed in vitro on purified DNA. Most of the 3-methyladenine was apparently released during the ten-minute period of alkylation treatment, the remaining 3-methyladenine being more slowly released from the DNA, but still at a rate much faster than that observed

117 TABLE

Excision

of ,?-methyladenine

Strain

BW9101 (tag+) (at, 30°C)

BK2012 (lag-2) (at 30°C)

PK432 (tag-l) (at 43°C)

from DXA in viva following dimethyl sulphate

Incubation time after alkylation (min) 0 5 15 30 0 5 15 30 0 5 30

5

hIolecules/cell ‘I-Methyl3.Methyladcnine guanine

528

28

454 427 417 451 469 493 405 506 545 486

17 14 13 76 I.5 74 61 83 90 x5

treatment with

Ratio 3.methyladeninei 7.methylguanine 0.052 0.036 0.033 0,027 0.169 0.159 0.151 0.150 0.163 0.165 0.17

Bacterial suspensions were treated with 0.56 mw[3H]dimethyl sulphate for 10 min at 30 or 43°C and subsequently incubated for various periods in minimal salts medium at the same temperature. Individual DNA preparations from each portion were hydrolysed and analysed for content of methylated purines.

for non-enzymatic hydrolysis. No detectable excision of 7-methylguanine was found. The results obtained with the two E. coli tug mutants BK2012 and PK432 after treatment with r3H]dimethyl sulphate were markedly different from those found with the tag+ strain. The DNA from the tug mutants contained about three times more 3-methyladenine than DNA from tug+ strains following alkylation ; the ratio of 3-methyladenine : 7-methylguanine was similar to that observed with alkylation DNA in vitro. Moreover, the level of 3-methyladenine in DNA remained high for at least 30 minutes. It is unclear if slow, active excision of a small proportion of the 3-methyladenine residues occurred in the BK2012 strain during this incubation. As with the tag+ strain, no detectable release of 7-methylguanine was observed. Similar results were obtained after treatment of bacteria with 75 PM-N-[methyL3H]methylN-nitrosourea; the BW9101 strain effectively removed 3-methyladenine residues from DNA, while little (or no) active excision occurred in the BK2012 strain (data not shown). In conclusion, these data show that 3-methyladenine-DNA glycosylase is essential for the rapid removal of 3-methyladenine from the DNA of 8. co&. The experiments above were carried out at low doses of dimethylsulphate that did not detectably impair cellular growth and allowed >95% survival of both E. coli tag+ and tag strains. Under these conditions, about 80 3-methyladenine residues per cell were present in the E. coli tag mutants (Table 5). Actively growing E. coli cells can apparently tolerate the presence of several 3-methyladenine residues per chromosome with minimal effect on survival. It is possible that a limited number of such lesions can be corrected by post-replication repair. In support of this notion, tag lexA double mutants have been found to be much more sensitive to alkylating agents than either tag or ZexA single mutants (G. B. Evensen & E. Seeberg, unpublished data).

,4t high levels of alkylation. however. the presence of large amounts of 3methyladenine in DKA appears to lead t,o inactivation and cell c1eat.h (Fig. 1). (i) Search for 3-methyludenirt,e resdues in the I),Xd oj’aSn H. coli tag strain that has twt been. exposed to alkylatin~g agents We observed that small numbers of 3methyladenine residues in t)he DNA of growing E. coli cells were tolerated without, signiticant rcduct)ion in survival (Table 5). It is conceivable that 3-mcthyladcnine residues may occur as minor. transient, cornponents of DNA during normal met.abolism and be removed l)y an enzyme such as 3-methyladenine-DNA glycosylase. If t,his w’ere the ease. taco mut,ants might be expect,ed to comain detectable amounts of such residues in their DNA. A11 att,empt to t,est this hypothesis was made hy growing E. coli BK2012 (tag) and it,s parent strain BW9101 (tag+) in medium containing [methyZ-3H] methionine. The naturally occurring and N6-mcthyladcnine. result from a methylat’ed DNA bases, i.e. 5methylcytosine post-replication event that involves enzymatic transfer of a methyl group directly from S-adenosyl mcthionine. and growth in the presence of radioactively labelled met.hionine results in selective labellinp of the minor methylated DXA bases. DXA was labelled, purified and hydrolysed. tirst under neutral conditions (pH 7.1. 1OCl”C. 30 min) to release any 3-mcthyladenine and subsequently under acidic renditions to release other purine residues. The amounts of radioactive 3methyladenine and X6-methyladenine were determined separately by paper chromatography. The DXA from both strain BW9101 (tag + ) and BK2012 (tag) was found to contain about 16,000 N6-methyladenine residues per cell (about 350,000 cts/min of N6-13H]methyladenirIc recovered per DKA preparation). a.nd this material accounted for more t,han 99;{, of the radioactively labelled purines. While the cells appeared to have a normal level of methplation (0.1 to 0.20,) at the N6 position of DNA adenine residues, little or no radioactive material co-chromatographed with 3methvladenine ((30 DNA residues, cell : < 1000 cts/min of 13H13-methyladenine recovered per DNA preparat,ion), and there was no detectable differenre between bhe tag+ and tag strains in this regard. While the present experiments do not exclude that one or a few 3methyladeninc residues may occur naturally in the E. coli chromosome, t’hey provide no clear evidcncc, for the presence of this hasc, and there was definitely no marked accumulation of such residues in the tag strain employed. (j) Spontaneous

mutation frequency

E. coli strains deficient in uracil-DNA glycosylase (ung) show a higher spontaneous mutation frequency than parental wild-type bacteria as a result’ of t’hc accumulation of deaminated cytosine residues which are not effectively removed in the ung cells (Duncan & Weiss, 1978). If 3-methyladenine occurs as a minor transient component of DNA during normal metabolism, tug mutants might also exhibit an increased spontaneous mutation frequency. However, no significant differences in spontaneous mutation frequencies were observed for tag+ and tag bacteria. (k) Genetic mapping

of the tag mutations

The tag-2 mutation in BK2012 (Hfr KL16) was transferred to the recipient AB1157 by conjugation, selecting for arg+ strR recombinants and scoring for the methyl

E. coli

TAG

119

MUTANTS

methanesulphonate-sensitive phenotype. To avoid inheritance of the xth marker from the donor, his recombinant5 were selected because xth is located close to his (White et al., 1976), and the his and xth+ characters of the recipient are likely to co-conjugate. The xth mutation was undesirable in the mapping studies because it confers some sensitivity to methyl methanesulphonate which could interfere in scoring of a tag mutation. One derivative, BK2106, was shown by enzyme analysis to be deficient in 3-methyladenine-DNA glycosylase; it contained a normal level of exonuclease III and t’hus had a tag xth+ genotype. This strain was used as recipient in crosses with various Hfr strains selecting for either MMSR (StrR) or His+ (St+) recombinants. A large number of MMSR recombinants (2.0% and 3.50/I, of recipient bacteria) were obtained in crosses with KL983 and PK191, 0.5% with KLl6, and less than 0.01% with KL209 and KL14. This suggested that the tag mutation was located close to the point of origin for chromosome transfer of KL983 and PK191, i.e. it would appear to be located between 43 and 51 minutes on the E. coli K12 map. The map position was determined more closely by scoring for MMSR among His+ (St+) recombinants (Table 6). A high percentage of MMSR recombinants was found in crosses with KL983, TABLE

Genetic mapping

Donor

KL16 KL983 KL96 PK191 AB259 KLF3/JC1552 BK2012 (tag-z) His + (SW) recombinants and tested for co-conjugation

qf tag

6

mutants by conjugation

BK2106 (tag-Z) as recipient Number of MMS* recombinants (%I tested 90 183 180 90

40 72 0 38

24

100

PK432.I (tag-I) as recipient Number of MMSa recombinants at 42°C ‘^” tested 22 22 54 24 22 48 91

were selected in crosses between the tag mutants of alkylation resistance.

86 86 94 83 50 100 5 and various

donors

PK191 and KL16, while none out of 180 tested was resistant among the His+ recombinants from crosses with KL96. This indicates that tag-2 is located between 46 and 51 minutes on the map. Good markers for transductional crosses in this region (see Fig. 6) are purF, n&A and n&A (Bachmann et al., 1976). Cotransduction with nalA was initially attempted, because nalidixic acid-resistant (NalAR) transductants can be selected directly from BK2106 or BK2012. The tag-2 mutation proved to be cotransducible with naZA at an observed frequency of 28% for BK2106 (Table 7). These frequencies were calculated from the number of MMSR recombinants found among NalAR colonies formed after Pl transduction. In experiments with BK2012, the number of transductants obtained on each plate was low, and an approximate cotransduction frequency of 15 to 30% was observed. Using Wu’s formula (Wu, 1966)

1’0

I’

li A It K A S

JC 7’ .-I I,

FIG. 6. E. coli K12 map showing tho positions of the tug mutations, origins of transfer of Hfr strains used in the mapping experirnent,s.

TABLE

Genetic

mapping

CIYXS

t Resistance

relevant

markers.

and the

7

of tag mutants by Pl transduction Selected phenotype

KK493

x BK2106

SalR

KK493

x BK2012

NalR

KK444

x BK2106

rialR

Nrd +

BK2107

x El01

KK444

x PK432.1

Nala

W3110

x PK43Z1

His+

Unselected phenotype MMSS MMSs MMSS MMSa MMSs MMSaNrd MMSsNrd MMSsNala MMSsNals MMSRNal” MMSaNals MMSst MMSs MMSal MMSs

Number of transductants

+ -

164 65 80 17 35 9 4 5 0 9 20 0 178 0 154

at 42°C.

it was calculated from the cotransduction frequency (28%) that tag-2 mapped 0% minute away from naZA. In order to determine the position of tag-2 relative to nalA, three-factor transductional crosses were performed with nalA, tag and nrdA (Table 7). A Pl lysate from strain KK444 (naZA nrdA) was used to transduce BK2106 to NalAR. Thirteen MMSR isolates were obtained from 35 transductants; nine of the

E. coli

!I”AG

MUTANTS

121

MMSR clones were found to be nrdA+ while four were nd4 -. These data indicate that tag-2 is located on the opposite side of naEA relative to n&A. Three-factor crosses between the same markers were also performed by transducing strain El01 (nrdA-) to r&A+ with a Pl lysate from BK2107 (naZA tag-Z). All of the tag-2 nrdA+ transductants were found to carry nalA, which confirms the relative gene order nrd-nalAtag. The tag-2 mutation is thus located at, 47.2 minutes on the E. coli K12 map (Bachmann et al., 1976). From their phenotypes, it was assumed that tag-l and tag-2 would be different mutations in the same gene. It was therefore expected that tag-l and nalA would be cotransducible. However, no cotransduction could be demonstrated between tag-l and nalA (Table 7). Conjugational crossest were then performed between various and scoring for the Hfr strains and PK432-1 selecting for His+ (StrR) recombinants temperature-dependent MMSS phenotype. In contrast to the results obtained with the tag-2 recipient, strain KL96 efficiently produced MMSR recombinants in crosses with PK432-1 suggesting that tag-l and tag-2 are mutations in two different genes located on each side of the origin for chromosome transfer of KL96, tag-2 being closer to his. A large number of MMSR recombinants were also obtained by mating PK432-1 with PK191 and the conjugational crosses place tag-l between 43 and 46 minutes on the E. coli K12 map (Table 6). This means that tag-l was expected to be with this marker. Cotransduction located sufficiently close to his to be cotransducible with h,is could not, however, be demonstrated (Table 7). The reason for this is not clear, but it is possible that tag-l is located in the very beginning of chromosome transfer for KL96, and that this site is too distant from his to give a detectable cotransductional frequency in our experiments. However, there could be other explanations for the mapping anomaly observed for the tag-l mutation in PK432 and further genetic analysis is required to solve this problem. The genetic mapping studies suggest that tag-l and tag-2 are mutations in different genes, a notion that is supported by tests for MMSR of His+ (StrR) recombinants formed in crosses between BK2012 (Hfr KL16, tag-Z) and PK432-1 (tag-l), which show that 5% of the recombinantx are MMSR at 42°C. The latter result is at least lOO-fold higher than would be expected from intragenic recombination (Low, 1973). Sexduction experiments showed by the F’ factor in KLF3 (Table 6) which that both tag-l and tag-2 are complemented confirms the position of tag-l derived from the conjugation experiments (Fig. 6). The gene location of tag-2 is beyond t’he map region reported for the F’ factor in KLF3 t The original PK432 is a slow growing strain which needs 24 hours of incubation at 30 to 42°C to give visible colonies on broth plates. This growth defect made it difficult to score the MMSS phenotype in ordinary plate tests, and a growth-proficient derivative was therefore constructed. Since the growth deficiency in PK432 was temperature-independent, and also because BK2012 (taq-2) had normal growth properties, it seemed unlikely that the 3.methyladenine-DNA glycosylase deficiency and growth deficiency in PK432 were caused by the same mutation. By conjugational crosses with various Hfr strains it appeared that the growth defect mapped at about 93 minutes on the E. co& map and thus could be a dnnB mutation. This was supported by the observation that, PK432 is unable to plate bacteriophage h which is characteristic of certain dnaB (qrof’) mutants (D’Ari et al., 1975). A short mating was performed between KL25 Hfr and PK432 selecting for Arg+ (Strs) recombinants. A growth proficient,, MMSs (at 42°C) recombinant was isolated (PK432-1) and assayed for 3-methyladenine-DNA glycosylase at 30°C and 42’C. This strain had the same thermolabile enzyme as the original PK432 strain and was used for mapping of the tag-l mutation. This strain had also regained the ability to plate bacteriophage /\.

(Low. 1972). although it has t)carn reportthd (Rlument~hul. 1!)72) that KLIJ3 c:ompll* merits m.ci%, and the ht,ter has hewn mapped b.v trnnschwtion ~~xp?rirn~:nt~sin :L fJOsiiiOli which appears to t)tb very close t,o tnq-2, It WARS possi 111~ that the, I<” factor in IiLIYi carries a small piece of t,he chromosomal rcpion around 47 minutes, hut has a d&tiotl in the 46 to 47 minute int,rrval (Fig. 6) \vhirh may represent a plasmid-mediatfld addition (see Discussion).

4. Discussion (a) 3-n~flthyladenin,e-L>NA glycosylase is re,spor&bEe for the removul 3-~methyladenine from DNA in vivo

qf

Lawley and co-workers (Lawley 8: Orr, 1970; Lawley & Warren, 1976) showed t,hat wild-type E. coli cells rapidly removed 3-methyladenine from their DNA following treatment with alkylating agent,s. An enzyme that releases 3-methyladenine from alkylated DNA by catalpsing the cleavage of the base-sugar bond of 3-methyl-dAMP residues was subsequently identified and purified from E. ~oli cell extracts (Lindahl 1976; Riazuddin & Lindahl, 1978). The present work describes two different E. roli mutants (tag) that are deficient in this enzyme activity. These mutants are greatly impaired in their ability to release 3-methyladenine from their DXA. We conclude that 3-methyladenine-DIVA glycosylase appears to be the enzyme responsible for the specific and rapid removal of 3-methyladenine from alkylated DNA in viva. In comparison to t,heir parent, strains, the two tag mutants are very sensitive to alkylating agents such as methyl methanesulphonatc (Fig. 1). On the other hand. the mutant,s exhibit, norma. survival afher exposure t,o ultraviolet light, ionizing radiation or nitrous acid. Similarly, these mutant.s are deficient in host cell reactivation of met’hyl methanesulphonate-treated bacteriophage X, but proficient in reactivation of ult,raviolet or X-irradiated phages. Several spontaneous revertants of one of t,he mutants have been isolated that are alkylation-resistant and have simultaneously regained normal levels of 3-methyladenine-DNA glycosylase activity, as determined by assays of cell extracts. These dat,a implicate 3-methyladenine as an inactivating lesion following treatment, of E. coli with alkylating agents. While the tag mutants are primarily killed by the presence of 3-methyladeninet in their DNA: it is not c1ea.r if this lesion is a predomina,nt cause of inactivation of wild-type cells t,hat have been exposed t’o high concentrations of agents such a,s methyl methanesulphonate. In a separate study, it has been observed that E. coli tag mutants also show greatly increased mutation frequency after treat~ment with alkylating agents, suggesting that 3-methyladenine is a premutagenic lesion (I. Mfsteng & E. Seeberg, unpublished results). It has been proposed by Lawley & Warren (1976) that the rapid removal of 3-methyladenine from the DNA of wild-type cells could reflect a mechanism that keeps the minor groove of the DNA helix free from groups which might interfere with the binding of polymerases and other essential enzymes of nucleic acid metabolism. The methyl groups of the naturally occurring minor DKA bases %methylcytosine and t A contribution of the very minor 1976) has not been excluded, although DNA glyconylase (Riazuddin & Lindahl,

alkylation this lesion 1978).

product 3.methylguanine is not released effectively

(Lawley C Warren, by 3.methyladenine-

E.

c-01;

TAG

hII.7TASTS

123

N6-methyladenine are located in the major groove, so the minor groove is normally free from such groups. In support of this hypothesis, which suggests an important role for 3-methyladenine-DNA glycosylase in preserving the integrity of DNA, promoter recognition by bacteriophage T7-induced RNA polymerase does appear to involve preferential contact with the minor groove of the DXA helix (Stahl & Chambarlin, 1978). On the other hand, recent, sequencing studies on the DNA regions that, are protected from chemical modification by bound E. coli RNA polymerase have shown that this enzyme recognises residues both in the major and minor groove (Johnsrud, 1978), so in this case there is no evidence for a selective interaction with the minor helical groove.

(b) Occurrence in DNA

of adenine residues suhatituted in the 3 position

Is the physiological role of 3-methyladenine-DNA glycosylase exclusively to remove 3-methpladenine present in DNA after cells have been exposed to alkylating agents? It may be argued that this could be the case since several different enzymes which appear to act specifically on alkylat’ion lesions in DNA have been found recently in cell-free extracts of E. coli. Thus. while 7-methylguanine appears innocuous in DNA in that it has the coding properties of unmodified guanine and is ignored by repair systems (Strauss 4 al., 1974), a secondary product, of this lesion, derived by slow hydrolyt’ic cleavage of t’he imidazole ring, is released in free form by a distinct DNA glycosylase (Chetsanga & Lindahl, 1979). Further, an inducible enzyme which participates in the repair of rY-methylguanine (Robins & Cairns, 1979; Karran et al., 1979) and appears to be the product of the E. coli ada gene (Jeggo, 1979) has been demonstrated. 3-Methyladenine is a major alkylation lesion, and a specific repair enzyme may similarly be required for its removal. N-Nitroso compounds are readily generat’ed by the reaction of nit’rite, obtained by bacterial reduction of nitrate, wit,h a wide variety of secondary amines (Lijinsky & Taylor, 1977). so alkylating agent,s may occur relatively frequently in the natural environment and provide a selective advantage for organisms with the ability to repair alkylation damage. Alternatively, the reason for the widespread occurrence of 3-methyladenine-DNA glycosylase (Lindahl, 1976: Laval, 1977 ; Brent, 1979 ; Ishiwata & Oikawa, 1979) might be that it is required to remove a, hypothet,ical lesion chemically similar to 3-methyladenine, such as an adenine N3-oxide residue, which might be generated by reaction of DNA with peroxides (Teller et al., 1978). The only N3-substitut,ed adenine known to occur naturally in cells that have not been treated with alkylating agents is triacanthine (6-amino-3-dimethylallylpurine). Although this compound is found in certain plan& it is not known whether it is also present in DNA. It is noteworthy that triacanthine and 3-ethyladenine have very similar crystal st)ructures, and these differ significantly from that of an unsubstituted adenine residue (Pet,ersen & Furberg, 1975; Kistenmacher et al., 1977). The tag mutants are strongly impaired in their ability to remove 3-methyladenine from their DNA. However, it should be noted that neither mut,ant is completely devoid of 3-methyladenine-DNA glycosylase activity in, vitro, since much smaller amounts of an apparently different: more heat-stable activity have been observed in extracts of RK2012, as well as in wild-type strains. Thus, the tag mutants may be able to remove small amounts of 3-methyladenine from their DNA in viva and conse-

quently. the absence of a detectable accumulation of 3.meth?rlatleninp in the I)XX ot a fng mutant during normal cell growth does not exclude the possibility that, it might OCCIX as a transient minor Dh’X base residue during normal rnct:II)olism In tna~w malian tissue, an S-adenosylmrthionine-requiring enzyme that catalyses tht methyl~rCon of free adenine t,o 3-met’hyladenine i,j ‘vitro has htvn described (Axelrod & Dal>,. 1962). On the other hand, 3-methyladenine has never been found a:: n normal constituent’of nucleic acids, nor does it seem to be one of the many modified baseh of transfer RNA (Gauss ct al., 1979). (c) Nap

locatio,ls of tag mutations

The tag-l and tag-2 mutations have been found by conjugational crosses to map in a relatively poorly defined region of the E. coli K12 chromosome around 46 minut’es on the revised map (Bachmann et al., 1976). The tag-2 mutat,ion has been subsequently mapped by three-factor transductional crosses at) 47.2 minutes (Table 7). The tag-l mutation (which is in the st’ructural gene for the enzyme) behaves somewhat differently and has been tentatively mapped bet’ween 43 and 46 minutes. Thus, tag-2 showed 28% cotransduction with &a, which is located at 47.9 minutes, while no cotransduction (
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to ultraviolet lighb and ionizing radiation has been isolated and partially characterised by Yamamoto and co-workers (Yamamoto et al., 1978; Yamamoto & Sekiguchi, alk, which has been mapped at 43 to 44 minutes, shows 18% 1979). This mutation, cotransduction with his and therefore seems to be located in a position different from tag-l and tag-Z. Crude cell extracts from the alk mutant and the parent strain were found to contain comparable levels of 3-methyladenine-DNA glycosylase activity (Yamamoto et al., 1978). The precise enzyme defect in the alk mutant has not been defined.
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Strauss, B., Scudiero, D. & Henderson, E. (1974). In &I o 1ecular Mechanisms for Repair of DNA (Hanawalt, P. C. & Setlow, R. B.. eds), part ,4, pp. 13-24, Plenum Press, New York. Sutherland, B. M. & Hausrath, S. G. (1979). J. Bacterial. 138,333-338. Teller, M. N., Giner-Sorolla, A., Stohrer, G., Budinger, J. M. & Brown, G. B. (1978). Cancer Res. 38, 2229-2232. Verly, W. G., Paquette, Y. & Thibodeau, L. (1973). Nature New Biol. 244, 67-69. wall, J. & Harriman, P. D. (1974). Virology, 59, 5322544. Weiss, B. (1976). .I. Biol. Chem. 251, 1896-1901. Whit,e, D. J., Hochhauser, S., Cintron, N. M. & Weiss, B. (1976). J. Bacterial. 126, 1082-1088. Wu, T. T. (1966). Genetics, 54, 405-410. Yajko. D. M. & Weiss, B. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 688-692. Yamamoto, Y. & Sekiguchi, M. (1979). Mol. Gen. Genet. 171, 251-256. Yamamot,o. Y., Katsuki, M., Sekiguchi, M. & Otsuji, N. (1978). J. Bacterial. 135, 144-152.