Human deoxycytidine kinase as a conditional mutator in Escherichia coli

0 Academic des sciences / Elsevier. Paris Biol@gh et g&G$que molk&ires / Molecular

biology

and genetics

Human deoxycytidine mutator in fscherichia

kinase as a conditional co/i

La dgsoxycytidine kinase humaine comme mutateur conditionnel chez Escherichia coli MADELEINE

BOUZON,

Groupe de cbimie 75015 Paris

biologique,

PHILIPPE MAKLI&E* unitPde

biocbimie

cell&ire,

CNRS

Ura 1123,

Institut

Pasteur,

28, rue du Docteur-Rokcu,

La diversification chimique des prtcurseurs de 1’ADN a et6 entreprise dam Escberichia coli en exprimant le gene de la dtsoxycytidine kinase humaine dans des bacttries cultivees en presence d’analogues de nucleosides qui varient par la base ou le sucre. Lorsqu’il y a expression du gene recombinant, l’arabinocytidine et la didesoxycytidine deviennent toxiques B des concentrations moindres que millimolaires. Les dtsoxynucleosides portant les bases ambigues isoadenine (Z-amino-purine) et isoguanine (2-hydroxy-G-amino-purine) provoquent une augmentation de la frequence de mutation comparable a celle obtenue en presence des plus puissants alleles mutateurs (dnaQ). Ces resultats ouvrent la voie vers la propagation d’acides nuclCiques chimiquement remanib et vers l’h ypermutagenese control& des plasmides in vivo. Mats

cl&

I mufugen&e,

Z-amino-purine,

isood&nine,

isoguanine,

biosynfh&e

de rADN

ABSTRACT The chemical

diver$cation

ofDMA

precursors

was u&rtaken

human

gene jr deoxycytidine kinase, and supplying analogues bearing an altered base or sugar. Arabinorytidine toxic to E. coli in thesub-millimolar range. Deoxynucleosides

suck

in

Escherichiacoli by

expressing

the

recombinant strains with nucleoside and dideoxycytidine thus became high& bearing isoadenine (2-aminopurine) and

isoguanine (2-bydroxy&aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that oftbe most e&ient mutator alleles (dnaQ). Thesefindings open the way to thepropagation chemically remodelled nucleic acid and to the controlled bypermutagenesis ofpkzsmids in viva.

of

Key words:

mutagenesis,

Z-amino-purine,

isoadenine,

isoguanine,

DNA

biosynthesis

VERSION ABRISGBE La varied desdesoxynucltosideset de leursanaloguesayant accb au pool desprecurseursphosphorylesde 1’ADN dans Escbericbia coli K12 a tte elargiepar expression du genede la dboxycytidine kinasehumaine, dont le cDNA, recemment clone, a et6 introduit dansle vecteur d’expressionpTrc99A.

Note

pr&enr&parMaurice

Note

remise

Plusieurs Ser.

le 13 f&tier,

Hofnung accept&e

erteuts d’impression ant L& 1997. 320, 207-214

III&i.

*Correspondence

Cet enzymesecaracterisepar sacapacitea phosphorylerune largegammede nucleosidesdiff&ant aussibien par le sucre que par la basepurique ou pyrimidique, a l’exception de la thymine. Lorsque la desoxycytidine kinase est exprimee, 1’a;abinocytidine et la didesoxycytidine, qui ne sont pas

aprhs

rkvision

le 17 f&et

altCrC cette note in ; elle esr ici republik

1997

C. R. Acad. Sri. in extenso.

and reprints

C. R. Acad. Sci. Paris, Sciences 1997, 320,427-434

de la vie / Life Sciences

427

M:Bouzon,

P. Marlihe

metabolistes par E. co/i sauvage, deviennent toxiques pour les batteries recombinantes avec des concentrations minimales inhibitrices de 40 PM et 160 pM respectivement. L’utilisation de la desoxycytidine kinase humaine comme mutateur conditionnel dans E. cob a ete testee en cultivant des batteries recombinantes en presence de nucltosides portant des bases ambigues, l’isoadenine (Z-amino-purine) et I’isoguanine (2hydroxy-6-amino-purine). Lorsque l’expression de la desoxycytidine kinase est induite, la frtquence des mutations de resistance & la valine augmente de I’ordre de 4 500 fois en presence de desoxyribosyl-isoadenine et de 800 fois en presence de desoxyribosyl-isoguanine, toutes deux a des concentrations de 30 g 100 pM. Ces resultats sont cornparables a ceux obtenus avec le plus efficace des alleles mutateurs connus dans E. coli, dnaQ::miniTnlO, dans les memes conditions experimentales. Mettant a profit cette nouvelle voie metabolique permettant a des nucleosides exogenes d’acceder a I’ADN chez E. co& nous avons etudit les mecanismes de mesappariement de l’isoadenine et de l’isoguanine dans des souches faux-sens construites par le laboratoire de J. Miller, dont le codon Glu461 du gene de la J3-galactosidase a ete remplace par d’autres codons, et telles que la survenue de chacurie des six substitutions possibles puisse &tre detectte par restauration du codon Glu necessaire a l’activite catalytique de l’enzyme. Ces souches, dans lesquelles le plasmide portant le gene de la dtsoxycytidine kinase humaine a ete introduit, ont ete cultivees en presence des deux analogues promutagenes et la frequence des reversions Lac+ a ete mesuree. Les resultats obtenus confirment le mtcanisme mutationnel de l’isoadenine par transition de G:C vers A:T et mettent en evidence l’induction par l’isoguanine de transversions de G:C vers T:A. Nous proposons un schema d’incorporation de l’isoguanine en configuration ~yn face a la guanine suivie de son appariement avec la thymine lors du prochain cycle de replication. L’activite desoxycytidine kinase introduite chez E. coli ouvre une voie metabolique inedite dans cet organisme. Elle catalyse la premiere &tape de l’activation des nucleosides naturels et permet l’acces au pool de monomeres de I’ADN a des analogues structuraux. El1 e f ournit un mutateur conditionnel aisement controlable qui pourrait permettre d’elaborer in vivo une alternative aux techniques d’hypermutagenese in vitro.

metabolic

pool

of

DNA

precursors

can

be

altered

either quantitatively by establishing concentration imbalances between the four deoxynucleoside triphosphates, or qualitatively by generating non canonical nucleotides in living cells [I]. Greater mastery than heretofore achieved is needed for(i) delivering very high plasmid mutation rates under controlled external conditions, and (ii) propagating episomes that differ chemically from DNA and RNA. Both these goals imply genetically reprogramming the catalysts involved in the metabolic pathways of DNA precursor a generic

428

minimum trend, we

number investigated

of

metabolic steps. the phosphorylation

Following this of deoxynu-

cleosides in Escherichia co/i K12 by human deoxycytidine kinase (DCK), an enzyme whose cDNA has been recently cloned and sequenced [2]. This target is especially promising because E. co/i lacks deoxynucleoside kinase activities except the [ok-encoded thymidine kinase which is highly human rylates

specific for the bases T and U [3]. In contrast, DCK displays much wider activity and phosphonucleosides with modified sugars such as arabi-

nose with DNA

or dideoxyribose and bearing purines the notable exception of T [4]. We metabolism of E. co/i can be enlarged

means and that its genome fic mutagenesis pathways, to that of the most powerful

Materials Culture

or pyrimidines report here that by this simple

can be targeted with an efficiency mutators.

through specicomparable

and methods

of bacterial

strains

Bacteria were routinely grown in rich Luria-Bertani medium (LB) prepared according to Miller [5] or in minimal medium supplemented with glucose 2 g/L as described previously [6]. Growth media were solidified with 15 g/L agar (Difco) for the preparation of the plates. Liquid and solid cultures were incubated at 37 “C. When required, antibiotics were added at the following concentrations if not stated otherwise: carbenicillin, 100 mg/L; tetracyclin, 15 mg/L. For the induction of gene expression

isopropyl-B-D-thiogalactoside

(IPTG)

was

added

at

0.5 mM. Strain The this

and

plasmid

construction

E. co/i K12 strains and the plasmids constructed study are listed in table I. The pDCK1 plasmid

for was

constructed by ligating the Ncol-BarnHI 780-bp fragment of the pET3d derivative carrying dck cDNA (described in [2] and obtained from Dr B.S. Mitchell) in Ncol-BarnHI digested pTrc99A vector (Pharmacia). The p7077 strain was obtained by two consecutive Pl transductions. MCI 655 was first infected with a Pl lysate from KU8 [7] in order to transfer the AserB deletion and the nearby

Introduction The

precursors thus appears to be a straightforward way to generate the widest variety of deviant deoxynucleoside triphosphates with an altered base or phosphosugar in a

biosynthesis. Evolving enzymes activity towards a whole class

endowed of nucleic

with acid

TnlO marker. Tetracyclin resistant clones were selected and tested for serine auxotrophy. One of these clones, B7071, was then infected with a Pl lysate from S0928 and serine prototrophs were selected on minimal medium plates. All selected Ser+ transductants carried the deletion of the deo operon as they were unable to grow in minimal medium with thymidine as only carbon source. Minimal assay The

MlCs

inhibitory

concentration

of the

by a published

nucleoside method

C. R. Acad.

[8].

(MIC)

determination

analogues

were

An overnight

Sci. Paris, Sciences

determined

bacterial

culture

de la vie / Life Sciences 1997.320,427-434

Human deoxycytidine Table I. Bacterial

strains

kinase

in E. co/i

and plasmids.

Strain

Relevant

MG1655

F‘ X-

KU8

trxB::TnIOKan

MS2131

dnaQ::minilnIO

Gift of J. Shapiro

S0928

Adeo A/at thi upp udp ton ara A(/ac proB) 13

Cupples

and Miller

[23]

cc1 01

genotype

Construction Gift of 6. Bachmann

hsert3 zjj::TnlO

F’ /acZ:Glu46lam

Uhland

et al. [7]

Gift of P. Nygaard

proB+

cc1 02

ara

A(/ac proB) 13 F’ /acZ:Glu461 Gly proB+

Cupples

and Miller

[23]

cc1 03

ara A(/ac proB)

Cupples

and Miller

1231

cc1 04

F’ lacZtGlu461 Cln proB* ara A(/ac proBlJ3

Cupples

and Miller

[23]

Cupples

and Miller

[23]

Cupples

and Miller

1231

13

F’ /acZ:Glu461 cc1 05

ara

Ala proB+

A(lac proB)

J3

F’ /acZ:Glu461Val

pro@

CC1 06

ara A(lac prot?) F’ /acZ:G]u461

13 Lys proB+

87033

F- hpTrc99A

p7034

F- hpDCK1

b/a+ /a&

p7071

AserB zjj::Tn

p7077

Adeo Adeo

P7086

pDCK1

Transformation

of MGI

655 with

Transformation

of MG1655

pTrc99A

b/a+ /a&

JO

b/a+ /a&

with

pDCK1

dck+ Transduction

of MG1655

Transduction

of p7071

Transformation

of p7077

with with

Pl lysate from

PI lysate from

with

pDCK1

dck+

p7203

ara A(/ac proBi J3 F’ /acZ:Giu461 am prof?+ pDCK1 b/a+ /a& dck+

Transformation

of CC101

with

pDCK1

p7204

ara A(/ac proB) 13 F’/acZ:Glu46IGlyproB+ pDCK1 b/a+ /a& dck+

Transformation

of CC1 02 with

pDCK1

p7205

ara A(/ac pro61 13 F’ /acZ:Glu461 Gin proB+ pDCK1 b/a+ /acP dck+

Transformation of CC103 with pDCKl

p7206

ara A(lac pros) 13 F’ /acZ:Glu46IAla proB+ pDCK1 b/a+ /a& dck+

Transformation

of CC1 04 with pDCK1

07207

ara A(/ac proBl J3 F’ /acZ:Glu461 Val pro8+ pDCK1 b/a+ Iace dck+

Transformation

of CC1 05 with

pDCK1

p7208

ara A(/ac proB) J3 F’ /acZ:Glu461 Lys pro/?+ pDCK1 b/a+ /a& dck+

Transformation

of CC1 06 with

pDCK1

p7232

dnaQ::miniTnI

Transduction

Plasmid

Markers

Construction

pET3d::dck pTrc99A pDCK1

bla+ dck+ b/a+ /a& bla* Ia&

Gift of B.S. Mitchell [2] ColEI replicon (Pharmacia) Cloning of dck from pET3d::dck

O:tet+

dckf

in minimal medium plus 2 g/L glucose and 25 mg/L carbenicillin was diluted 1 OO-fold in the same medium and cultivated at 37 “C with aeration until a turbidity of about C. R. Acad. Sci. Paris, Sciences 1997. 320.427-434

KU8

S0928

de la vie / Life Sciences

of MG I 655 with

Pl lysate from

MS2 13 1

in pTrc99A

0.1 O.D. (600 nm) was reached. The culture was then diluted 2.5fold and distributed to a 2-fold serial dilution of a nucleoside analogue with and without IPTG. After an

429

M. Bouzon.

P. MarWe

18 h incubation mined as the turbidity was

at 37 “C with lowest analogue detectable.

aeration the MIC was deterconcentration for which no

ted in pDCK1,

Materials

nucleoside analogue with and without IPTG. The cells were grown for 18 h at 37 “C before plating. Viable cell counts were determined on LB plates. Valine resistant cells were selected on minimal medium plates containing

over

viable

of

Lactobacilus

nucleoside

2’,3’-dideoDeoxyisoausing

N-deoxyribosyltransferase according to the

leichmannii

bofuranosyl)imidazole-4-carboxamide ted elsewhere (Dr. S. Pochet,

pers.

a

Wild

type

of nucleotide kinase strains

of E.

pools

by human

co/i K12 are refractory

to antimeta-

data DNA

variety of deoxynucleotide by expression of a foreign The strain B7034 produces plasmid carrying a cDNA

Activation

Table

II. DCK-mediated

toxicity

Strain

could

be enlarged kinase gene. kinase from a enzyme as isola-

of deoxynucleoside

sugar

analogues

warrant monomer

use of the human dck gene biosynthesis in E. co/i.

of promutagenic

The human DCK various nucleosides

toward

Minimal

p7033

pTrc99A

> 2500

p7034

pDCK1

> 2500

inhibitory

concentrations,

expressed

in FM,

were

added

enzyme differing

to the growth

is known widely

to phosphorylate in their nucleobase

medium ddC

with

/PTC

without

> 2500 40 assessed

diversifying

nucleosides

araC IPTC

for

F. co/i.

Drug

Genotype

without

of chain

ves comparable to those of the canonical DNA precursors are formed via DCK in our recombinant strain. Although this point was not investigated quantitatively the genetic

bolites mimicking deoxynucleosides of the bases A, C and G, because they contain no enzyme able to phosphorylate such compounds. We investigated whether the metabolites deoxynucleoside deoxycytidine of the human

to very low amounts for 3’-azido-3’-deoxythymi-

dC sugar-analogues also implies that the subsequent phosphorylation steps of their monophosphates occur in E. co/i cells. These metabolic steps are presumably catalysed by cmk or pyrH-encoded nucleoside monophosphate kinases and ndk-encoded nucleoside diphosphate kinase. The low MIC values which we obtained for araC and ddC suggest that pools of their triphosphate derivati-

be repor-

Results and discussion Diversification deoxycytidine

of the former DNA strands,

dine (AZT) [12]. By comparison, araCMP can be further elongated albeit at a very reduced rate after its incorporation by DNA polymerases [13, 141. The toxicity of the two

(2-

[I 01 will comm.).

that incorporation to further elongate

thesis in E. co/i is vulnerable terminators, as demonstrated

reported

procedure [9]. The synthesis of deoxyisoguanosine hydroxy-6-amino-9-(2’-deoxy-P-D-ribofuranosyl)purine) prepared by the ring closure of 5-amino-(2’-deoxy-P-D-ri-

in (see was

and that incorporation of the latter can be either reversed by nucleolytic action or pursued by condensation of further deoxynucleoside triphosphates. Addition of deoxycytidine at 160 pM alleviated toxicity of the two analogues (data not shown). It has been shown previously that ddC causes DNA chain termination in human cells after its phosphorylation by DCK [l I]. In a similar way, DNA syn-

Chemicals

extract

This plasmid, original DCK

of a lactose repressible operator encoding the lactose repressor Lacl methods). The recombinant strain

respectively, suggests results in the inability

cells.

Cytosine-1 -P-D-arabinofuranoside (araC) and xycytidine (ddC) were purchased from Sigma. denosine (2-amino-9-(2’-deoxy-P-D-ribofuranosyl)purine) was prepared by enzymatic transglycosylation

[21. the

teriostasis (data not shown). These effects can be explained by the condensation of their triphosphate derivatives at the elongating 3’ end of DNA. The contrast between the bactericidal and bacteriostatic action of ddC and araC,

80 mg/L DL-valine. Lac+ revertants were selected on minimal medium plate containing 0.2% lactose as carbon source. The mutation frequency was defined as the ratio cells

and

laboratory by placing

highly sensitive to the nucleoside analogue arabinocytidine (araC) and to a lesser extent also to dideoxycytidine (ddC) when the lactose inducer IPTG was added to the growth medium (table II). The compound ddC was found to be bactericidal for p7034 at 640 pM concentration whereas treatment with 160 ~JM araC only resulted in bac-

The cells to be treated were cultivated overnight in minimal medium plus 2 g/L glucose and 25 mg/L carbenicillin. The culture was then diluted 1 04-fold in the same medium and mixed with a lo-fold concentrated solution of the

of mutant

Mitchell’s constructed

cDNA downstream pTrc99A, a vector

Mutagenesis

crude from

Beverly was

as described

in Ref.

[81. Each

C. R. Acad.

/PTG

with

/PTG

> 2500

> 2500

> 2500

160

experiment

Sci. Paris,

was

Sciences

performed

de

twice.

la vie / Life Sciences 1997.320,427-434

Human

deoxycytidine

kinase

in E. co/i

IPTG and diA or diG suggested that phosphorylation of these nucleosides by DCK was directly involved in the mutagenic process. An alternative mutagenic pathway would consist in the release of the purine base analogues iA or iG by purine nucleoside phosphorylase (deoD gene product) prior to their incorporation into the purine ribonucleotide pool by phosphoribosyltransferases and their subsequent conversion in DNA monomers via ribonucleotide reductase. Similar mutation in the presence of the promutagenic gues and IPTG with the strain 07086 catabolize deletion that the 20

0

40

Nucleoside Figure 1. LXX-mediated side base analogues.

60

analogue

mutagenesis

ValR mutant frequencies in the presence of

80

were determined IPTG for increasing

(FM)

with

deoxynucleo-

in p7034 cells ConcentraGons

because of a deo operon The results altogether indicated salvage of iA and iG had a negli-

gible contribution to the mutagenic pathway ponding deoxynucleosides. The production presence of exogenous diA at 30-I 00 pM

100

concentration

of E. coli

deoxynucleosides (data not shown). DeoD-mediated

cultivated of diA

(squares) or diC (circles). Each point corresponds to the mean result of three independent cultures. Bars represent standard errbrs. The background ValR mutant frequency in the absence of expression of dck or of base analogues amounted to 0.46 + 0.15 x lo-'.

accepting

both

purines

in with

E. co/i was ambiguous

its mutagenic potential employing nucleosides The

deoxynucleoside

hydroxy-6-aminopurine, denosine (diA) vely, were mutagenesis

derivatives

chosen as targets. by 2-aminopurine

encoded

proofreading

subunit

diATP

of E. co/i strains not inhibitory shown). increased

However, when

addition

of 100

results assayed shown sensitive

the dck

FM

is believed [15, 161 and group pair

as the purine [I 7, 181 and

the dck gene, while of dck expression

of representative by the frequency in figure 1. One

and

800-fold

experiments of valine recalls that valine

diG (data

was not

of E. co/i 4 500-fold

was by

by 100

pM

results were medium.

close gene

to the background was repressed

mutation

rates

were

C. R. Acad. Sci. 1997. 320,427-434

Paris,

The

of mutagenesis as resistant mutants are E. co/i K12 is naturally

because

of a genetic

in an acetohydroxyacid synthase [I 91. A linear ponse relationship to increasing concentrations nucleoside analogues could be reproducibly These growth

diG.

lesion

dose-resof deoxyobserved.

obtained in the presence of IPTG in the The mutation frequencies remained level when in the absence obtained Sciences

only de

la vie

expression of IPTG. in the presence / Life

Sciences

of the dck That high of both

III

DCK

and

frequency

(x 10’1 Strain

2-

to mediate diGTP has

mutator

ValR mutation

Drug

Genotype

added

to the growth medium

deoxyisoarespecti-

(diG),

mutation frequency was expressed about

diA

to exogenous

and

polymerase

of the conditional

[4].

thymine in addition to isocytosine was found to be a slight inhibitor

expressing regardless

E of DNA

[21 I.

investigated by pairing abilities.

designated

been elaborated by Steven Benner’s partner of an alternative Watson-Crick found to interact with [I 71. The diA nucleoside

pyrimidines

of 2-aminopurine

thereafter deoxyisoguanosine

and

and

of the corresof DCK in the concentrations

caused amplification of ValR mutation frequencies up to 4500-fold (table III). This level was comparable to that elicited by dnaQ alleles, the most efficient mutators known in E. co/i [20], when subjected to the same assay (strain p7232 in table Ill). Such mutator strains lack the dnaQ-

Table Ill. Comparative potency of a constitutive mutator.

component,

rates were obtained nucleoside analowhich is unable to

none MC1

655

wild

type

p7232

dnaQ::miniTnlO

p7034

pDCK1

b/a+

Each value corresponds cultures; standard errors

Miscoding

/aclQ

and

0.19

i 0.08

2180

i

101

0.46*0.15

dck+

to the mean are indicated.

by isoadenine

diA

result of three nd: not done

30 pM nd nd

22OOt800 independent

isoguanine

Taking advantage of the expeditious metabolic route from exogenous nucleosides to DNA which we implanted by expressing the dck gene in E. co/i, we investigated the precise miscoding Jeffrey Miller’s array of missense

pathways laboratory mutations

of iA and iG in this organism. has elaborated an exhaustive at codon Glu461 of /acZ that

inactivate the catalytic site of P-galactosidase in E. co/i [22, 231. Six such strains allow the assay of the reversion of the six possible types of base pair substitutions, by selecting for the restoration of Glu461 in P-galactosidase [23]. The results of reversion assays conducted with these six strains transformed with the &k-bearing plasmid pDCK1 are shown in figure 2. A IOthe number of La& colonies was

to 30-fold observed

increase in by treating

431

M. Bouzon,

P. MarHere

60

1

Figure 2. Reversion frequencies of Lac- strains induced by DCKactivated deoxynucleoside promutagens. White bars: no promutagen; hatched bars: diA 30 pM; so/id bars: diC 30 pM. Each number shown corresponds to the average value of two independent experiments.

0

Substitutiondetected Codon

the LacZ:E461G pared to other

from and

tion frequency P9OC genetic

mutants frequency

R7204

p7204 mutants.

diG of the

resulted LacZ:E461

A:T->T:A

Gln

Ala

Val

I37205

R7206

opposite opposite

of p7034 [231. further. Treatment

shown’in

figure

3. In the

in the to the

in an increase of the reversion A mutant p7206 by a factor of

following

round

this cal

non-canonical bases [17,

shown porated,

the

of the as

of replication

and transcription processes feasibility of information

purine together 181. Kamiya and

that iG pairs mainly albeit at a lower

with the collaborators

with T but rate, opposite

template sequence-dependent results corroborate the pairing

fashion of iG with

potential of diG vivo by promoting

purposes transversions,

432

for

mutagenic G:C to TA

T

in vitro, transfer by four

canonihave also

can also be incorC, A and G in a [24, 251. Our G and stress the in vitro thus

LYS

the

137208

effect

of the

mutM

and

mutY

mutator

alleles

of

shown

to

[26].

C in iA in

iG, now in the template strand, would have commanded incorporation of dTMP (figure 3). Benner and collaborators have previously studied the mispairing of iG and during replication and questioned

ling

We did not of the /acZ

about 50. This effect can be explained if the G base template paired with diGTP in syn configuration,

A:T->G:C

R7207

E. co/i

of replication, as rate of the muta-

about lOO-fold lower of p7204 as compared

genetic background this discrepancy by

of diAMP of dTMP

the following round 3. The amplification was

CC->T:A

with 30 pM diA as comThis can be interpreted

incorporation by incorporation

by diA background

G:C->C:G

GUY

67203

mutant missense

the template during depicted in figure

MC1655 investigate

amber

461

Strain

as resulting the template

G:C->A:T

A:T->C:G

and in paralle-

Conclusion The

mere

introduction

of a foreign

gene

was

enlarge the set of monomers that can enter DNA biosynthesis, and to provide a conditional mutator in the bacterium E. co/i K12. The mutator character is expressed only in the presence of exogenous synthetic nucleosides, with an efficiency comparable to that of the most powerful mutator genes. This permits finer control of mutagenesis than with constitutive mutators, and should allow the elaboration of an in vivo alternative to the hypermutagenesis techniques developed in vitro [27]. The scope of both in vitro and in vivo hypermutagenesis methods will now depend upon the diversification of ambiguous deoxynucleosides and of their pairing patterns [28, 291. The fact that an enzyme such as DCK, which has supposedly been improved by natural selection, is endowed with a wide activity toward abnormal nucleosides, suggests that human cells are not often confronted with such harmful compounds generated by foreign organisms or by indigenous aberrant metabolic reactions. Thus, the recombinant strains which we have constructed provide a model of human genotoxicity with which a variety of nucleoside analogues can now be conveniently assessed for their C. R. Acad.

Sci. Paris, Sciences

de

la vie / Life Sciences 1997.320.427-434

Human

deoxycytidine

[E-l

(C:GJ A

A C:iA

C:G

C : G

iG(syn)

A iG : T

A mutagenic base

pathways

T: iA

iG:T

c

analogues

C:G

A

IT:A]

3. Proposed

: G

A T : iA

C:G

of the deoxynucleoside diA and diC.

in E. co/i

B

A

figure

kinase

(A:TI

D

A. Incorporation of isoadenine. The top boxed base pair indicates the originalpair and the bottom one the resulting mutant pair. 8. lncofporation of isoguanine.

H

C. Pairing scheme of isoadenine with cytosine and thymine as implied in A. D. Pairing scheme of isoguanine with guanine and thymine as implied in B. An

iC(anti):Gfsyn) bonds

could

also

pair associatedby be drawn.

two

H-

iG(syn) : G

iA : C

,

iG : T

iA : T hazards as well as for their chemotherapeutic potential. Indeed, one recalls that human DCK mediates the toxicity of anticancerous drugs such as araC [30]. Finally, the very Aknowledgements. We thank Professors Michel Goldberg and We also thank Drs Beverly Mitchell for the gift of the pET3d::dck James Shapiro for the gift of strains, Sylvie Pochet for synthesis structions and Simon Wain-Hobson for improving the manuscript. cherche Medicale fellowship.

2. Chottiner D.. Ginsburg

T.A. 1992. DNA

Replication.

W.H. Freeman,

E.G., Shewach D.S., Datta N.S., Ashcraft E.. Gribbin D.. Fox I.H., Mitchell B.S. 1991. Cloning and expression

C. R. Acad. Sci. Paris, Sciences 1997. 320,427-434

de la vie / Life Sciences

DCK by the mutator to open DNA metamimics.

Maxime Schwartz for their constant interest and encouragement. plasmid, Barbara Bachmann, Per Nygaard, Michael Ehrmann and of the nucleoside analogues, Marion Mohr for initial plasmid conM. Bouzon was recipient of a SIDACTION-Fondation pour la Re-

of human deoxycytidine kinase cDNA. 88. 1531-1535 3. Munch-Petersen A. 1983. Metabolism

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