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Thèse de doctorat/ PhD Thesis Citation APA:

Villani, G. (1978, July). Les rôles des DNA polymérases dans la mutagénèse induite (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté des sciences, Bruxelles.

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Laboratoire de Biophysique et de Radiobiologie

Les r ôles des D N A polymerases dans la mutagénèse induite

Thèse présentée pour l'obtention du grade scientifique de Docteur en Sciences

( Section Biologie Moléculaire ) Giuseppe V I L L A N I

Juillet 1978

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UNIVERSITE LIBRE DE BRUXELLES

FACULTE DES SCIENCES

Laboratoire de Biophysique et de Radiobiologie

Les rôles des D N A polymerases dans la mutagénèse induite

Thèse présentée pour l'obtention du grade scientifique de Docteur en Sciences

( Section Biologie Moléculaire ) Giuseppe V I L L A N I

Juillet 1978

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rue des chevaux 67 - 1640 rhode-saint-genèse tél. 02/58.35.30

THESE ANNEXE

Il existe un contrôle hormonal de la croissance et de la différentiation des disques immaginaux dans la drosophlle.

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à Martine

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des idées et pour de nombreuses discussions ; Serge BOITEUX et Silvio SPADARI, pour leur participation active aux expériences et aux discussions ; • . •

et aussi

Geneviève MAENHAUT-MICHEL, pour sa collaboration dans la préparation du matériel expérimental et dans les discussions ;

Silvano GREGOLI, pour avoir soutenu l'émigrant au début et pour avoir exercé un esprit critique constant ensuite ;

Perrine CAILLET-FAUQUET, pour sa participation à la finition de cette thèse ; .

Oliver DOUBLEDAY, pour les discussions et les "anglifications"

des articles ;

Edmond, Joseph, Roland, Freddy et Percy, pour leur assistance techniqu Marie—Jeanne, Claudine et Tatiana, pour leur gentillesse et leur

efficacité ;

France, Christiane, Annick, Martial, Jan, Giovanni et Anne, pour leur aide amicale.

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GENERAL INDEX

Introductory Remarks on DNA polymerases

INTRODUCTION I : short introduction to genetics and biochemistry of mutagenesis

INTRODUCTION II : gênerai survey on DNA polymerases and their properties

Expérimental strategy and goals of the work Results

Discussion Conclusion Bibliography

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a DNA chain.

Such enzymes are involved in DNA replication (which Is an

accurate duplication of entire chromosomes and génomes), DNA repair synthesis (which is a gap filling or replacement of nucleotides excised in the course of the excision of DNA damages) and probably in some mode of genetic recombination e.g. providlng the strand displacement ( displacing one parental strand by _de novo synthesis on its complementary strand).

Genetics and enzymology of DNA polymerases gave support to the expected rôle of thèse enzymes in the accuracy of DNA replication ,:

i.e. in determining the mutation rates.

This thesis represents an in vitro' study on the rôle of DNA polymerases in induced mutagenesis , a study stimulated by the

genetics of induced mutagenesis which, in a stepwise fashion, leads us to suspect a genetic program for the tuning of the fidelity of DNA replication.

Introduction I is a short review of the genetics and biochemistry of DNA repair as related to mutagenesis, while Introduction II is a survey of DNA polymerases and their biological rôles.

/' 7 ''î «5 (>

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2.

Fig. 1. illustrâtes the essentiel polymerization reaction that is catalyzed by a DNA polymerase

INITIATOR

I I I I

A G c A T C G T I 1 I I

T T T A c T T G A C

M i l TEMPLATE

3'OH A T G

I I I T A C T C

dATP dCTP dGTP dTTP

I 1 I 1 1 I I "T"

A G C A A C T G T C G T T G A C

I I I I I I I I

•3 OH A T G C C

I I T C

_L_L

+ PPi ELONGATED CHAIN

Fig. 1 from Mildvan (la)

Note that only complementary deoxyribonucleotlde trlphosphates (dNTPs) can be used as monomer substrates which can be terminally incorporated only into a preexisting template-primer complex ;

"template" applies to the DNA chain that furnishes instruction for the séquence of nucleotides while "primer" is the segment of complementary DNA bearing a correctly paired 3*OH terminal nucleo- tide.

Divalent cations Mg^^ and Mn^^ are required for the reaction, probably in order to establish the correct conformation of the incoming dNTP (la).

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dNMP and the 3'OH terminal nucleotide of the primer.

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I N T R O D U C T I O N ,ï

- Short introduction to qenetlcs and blochemlstry of mutagenesis -

There appears to be 2 major pathways responsible for mutagenesis in E. coli :

" Direct mutagenesisy provoked by subtle modifications of DNA template or incorporated precursors and "indirect mutagenesis',' provoked by non- pairing DNA lésions which inhibit DNA synthesis.

Direct mutagenesis can be due to tautomers or isomers of normal bases, to incorporation of base analogues, to deaminating agents or some alkylating agents. Obviously, most of the spontaneous base change mutagenesis is direct mutagenesis by incorporation of non-complementar bases (see réf. 1 for a comprehensive review).

This mutagenic pathway is subject to genetic controls that include the activities of DNA polymerases (sélection of deoxynucleoside triphos- phates and the removal of incorrect ly inserted nucleotides ; see introduction II), perhaps some other proteins of the replication complex and also a post-replicative mismatch correction mechanism analogous to gene conversion (2, 3, 4). The error-avoidance function of an excision repair mechanism for the removal of mismatched bases nécessitâtes the existence of a discriminatory mechanism by which the parental (i.e. correct) and the newly synthetized strands (i.e. con- taining replication errors) can be differentiated. Generalized methylation has been suggested to provide the means for strand dis- crimination (2) and évidence supporting this hypothesis has been recently obtained (3, 4).

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Indirect mutagenesls Is due to usually bulky damages In the DNA which provoke a blockage of the elongation of new DNA chalns, l.e. thèse lésions cannot be copied by the normal DNA replication machinery.

Ultraviolet and ionizing radiation and many chemical mutagens and carcinogens, such as mitomycin C, activated aflatoxin and

benzo(a)pyrene cause such lésions.

Thèse DNA damages, when not repaired by excision repair, are believed to be responsible for the Induction in bacteria of a coordinately regulated group of functions, including a mutagenic DNA repair acti- vity known as "SOS repair activity" (5, 6). UV-induced cyclobutyl- pyrimidine dimers are the best studied lésions of this type and have been identified as a major cause of lethal (7) mutagenic (8) and tumorigenic effects (9) in a wide spectrum of organisms,

Wlld-type strains of E.coli effectively neutralize the potentially lethal effects of as many as a thousand pyrimidine dimers by utilizinç one or more of the three types of enzymatic DNA repair which are

schematically represented in Fig. 2.

Enzymatic photoreactivation is an error free repair process as is the majority of (short patch) excision repair, although mutagenic (long patch) excision repair may occur under certain circumstances (10).

Pyrimidine dimers which have not been removed from bacterial DNA by photoreactivation or excision repair, block DNA chain elongation but do not prevent subséquent reinitiation of DNA synthesis (11, discus- sion ) .

The majority of the resulting daughter strand DNA gaps are repaired by post-replicative recombination repair, (part C of Fig.2) which appears to be essentially error free ; however a minority of thèse gaps are repaired by the inducible "SOS repair" probably as the

resuit of error prone gap-filling DNA synthesis. This error prone DNA

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6.

repair, which may not involve DNA recombination (12) bas been suggested to be responsible for the raajority of UV-induced mutagenesis in E.coli.

(13).

Fig.2.

P H O T O R E A C T I V A T I O N E X C I S I O N R E P A I R P O S T R E P L I C A T I O N REPAIR

pynmidine f / dimer

photorealiyoting enzyme Cundî dimer

. [ X L

pholoreaclrvolinq liqhl

/ \ I diiner

splil

e n z yme reieosed

i n l o c t D N A

• r e p l i C Q l i o n

(dimers splil)

("short patch" pothwoy) UV \

incision by correndonucleose D

5 -3 -

excisionond repoir

replicotion by DNA polymerose I

1

sealmq by polynu- cleolide ligose

repaiied

DNA

replicolion

(dimeis removed)

3

(recombinalionol)

UV i

3 - . - 5!

3 —

replicolion

1 V

(disconlmuous dojghier suonds) recombinalionol exchonges by recombinotion enzymes

._/^^ .

O- y 3

repoif repliCQtiûn ond iigoM sealing

3- V

(continuous daughier slronds)

i

replicolion (dimers still présent)

Fig 2. Adapted from WITKIN (6)

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In the case of phage mutagenesis it has , been found that ultra- violet light irradiation of A and ^ X l 7 4 phages does not cause a significant increase in phage mutation frequency (14, 15, 16, 17) while irradiation of the host cell increases the frequency of muta- tions in both intact and UV irradiated phages (18, 19). This pheno- menon, which has been termed Weigle réactivation or W-reactivation (5) suggests that "indirect mutagenesis" occurs by the induction of a

transitory error prone DNA repair process (19). The induction of new E.coli gene functions required for the mutagenic DNA repair is further indicated by the observation that chloramphenicol inhibits this muta- genic repair (19).

Induced mutagenesis in bacteria dependsupon intact recA and lexA gènes, which is also true for )^ prophage induction (6).

Several évidences (6) show a complexity in the functions of recA and lexA gènes, whose products are required for the expression of a number of phenomena which are induced by "bulky DNA lésions" i.e.

"indirect mutagenesis"J inhibition of respiration (20), inhibition of cell division (21), induction of an inhibitor of exonuclease V (22), induction of protein X (23). Structural complexity of the two gènes is also inferred by the existence of several types of mutations : tif-1 (24) zab (25) lexB (26) tsl_ (27) spr (28) rnm (29). Thèse mu- tations were located tightly linked to recA and lexA and they cause modifications in the pleotropic effects of recA and lexA mutations.

Recently, a mutant called umuC was isolated (30, 31) which shows a decreased mutagenesis and a decreased Weigle réactivation without affecting either the inducibility of prophage X rio^ the inhibition of cell division following UV irradiation.

Of a particular interest seems the discovery of the so called

"protein X" (23), which is induced in large quantity in wild type cells by UV, nalidixic acid and several other inducing treatments and in tif-1 strain at 42°, since this protein has now been showed to be the product of the recA gene of E.coli (31a,31b) •

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8

Furthermore, induction of prophage A by tif^1 expression results in a lexA and recA dependend cleavage of the repressor (31c)and a

proteolytic activity was found to be a.ssociated with the X(recA) protein in tif 1 mutant (31d).

Indirect évidence is now available on the involvement of a protease activity (31e)in the induction of "SOS repair" (see article 5 of

discussion).

In the attempt to study the biochemical mechanism of this inducibl mutagenic DNA repair Radman and his collaborators (3if)took advantage from the fact that single stranded phage 174 appears to be subject to SOS repair (3ig 17, 31h).Since neither excision nor recombination repair can act on single-stranded DNA molécules, thèse two repair mechanisms are unlikely to be responsible for SOS repair. Furthermore, genetic évidence suggests that UV induced mutations of ç^/ 174 phage are producéd in the irradiated host cells only during the first round of DNA synthesis ; that is the passage from the single-stranded to double-stranded form of 174 DNA (17). Caillet-Fauquet et al. (3lf) have followed this transition from single-stranded to double-stranded DNA, using UV irradiated ^^4 phage infecting either intact or irra- diated host cells. They found that a single photolesion in d;( 174 phage DNA is sufficient to block completely DNA synthesis in intact host.cel]

(31f, 32).

However, when SOS repair was induced by UV irradiation of the host cel]

continuation of DNA synthesis past photolesions could be observed.

This, process probably involves incorporation of non complementary nucleotides (mutagenesis) and could be accomplished either through an induced decrease in the fidelity of the constitutive DNA polymerasei or through the induction of a new "error prone" enzyme (see introduc- tion II).

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Welgle-reactivation of DNA viruses has been reported to occur in human (KD, and some xeroderma pigmentosum) (33), rat (a tumor line) (34) and monkey (CV-1) cells (35,35).

Further évidence for inducibility of DNA repair processes in mammalian cells has been presented (37). In addition,expérimenta using the thymidine Kinase locus of Herpès simplex virus 1 (HSV-1) have shown that this Weigle-reactivàtion-like phenomenon is both

inducible and mutagenic (38).

This finding strongly supports the hypothesis that SOS repair and mutagenesis also occur in mammalian cells.

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10.

I N T R O D U C T I O N II

GENERAL SURVEY OF DNA POLYMERASES AND THEIR PROPERTIES

PROKARYOTIC DNA POLYMERASES

General properties

An enzyme that catalyzes the growth of a DNA chain is called a DNA polymerase. DNA polymerases have been found in extracts of ail cells, bacterial, plant and animal, where DNA synthesis has been measured. The DNA polymerases of E.coli are designated pol I, pol II and pol III in the order of their discovery (39, 40, 41). Récognition of this séries of enzymes in E.coli led to comparable genetic and bio Chemical studies in Bacillus subtilis and the discovery of a similar pattern (42, 43). The DNA polymerase isolated from Micrococcus luteus an organism known earlier as M. lysodeikticus, resembles pol I (44, 45, 46).

Table I shows some properties of E. coli DNA polymerases :

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Properties of Polymerases I, II and III of E. coll

Pol I Pol II Pol III Functions

Polymerization: 5' 3' Exonuclease: 3' 5' Exonuclease: 5' 3' Template-primer:

Intact duplex

Primed single strands

Nicked duplex (poly d(A-T))

Duplex with gaps or protruding single- strand

5' ends of: < 1 0 0 nucleotides

> 1 0 0 nucleotides Polymer synthesis de novo

Activity;

Effect of sait:

percent of optimal

20 mM KCl 50 mM KCl 100 mM KCl 150 mM KCl Effect of 10 percent ethanol,

percent of optimal Km for triphosphate

Inhibition by arabinosyl CTP

Inhibition by sulfhydryl (-SH)-blocking agents

Lability at 37°

Inhibition by pol I antiserum,

+ + +

+ +

+ + +

60 80 100 80

40 low

+

• +

60 100 70 50

45 low +

+ + + •

100 50 10 0

200 high

•+

+

General :

Size, daltons

Affinity to phosphocellulose : molarity of phosphate required

for elution Molécules/cell,estimated

Turnover number , estimated Structural gènes

Conditional lethal mutant

109.000 120.000

0.15 M 400

(1) pol A

yes

0.25 M 100 0.05 pol B

no

180.000

0.10 M 10 15 dna E yes (from DNA synthesis by A.Kornberg - Freeman Ed. San Francisco) D.M. Livingston and C. Richardson JBC 250 470 (1975)

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12.

E.coli DNA polymerase I can be cleaved in two sub-components ; a large fragment (76,000 daltons) containing the polymerase and the 3'-^ 5' exonuclease activity and a smaller one (36,000 daltons) with only the 5'-^ 3' exonuclease activity.

The large fragment may be obtained in two ways : by cleavage of intact enzyme with subtilisin, trypsin or other proteases (47) ; or by isolation from E.coli extracts, presumably a product of proteolysis (48).

The DNA polymerases of E.coli-bacteriophages T^ (49) T^ (50), (51) and of B. Subtilis-bacteriophage SPO^ (52)have been also extensively studied. Ail thèse bacteriophages seem to possess a unique form of DNA polymerase.

Table II shows a comparison between E.coli Pol I and the phages T , T , T and SPO induced polymerases.

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Comparlson of pol I and the phage T4, T5, T7, and SPOl-lnduced polymerases Pol 1 T'4 T5 T7 SPOl

Functions:

Polymérisation: 5' 3' + + + +

Exonuclease: 3' 5' + + +

Exonuclease: 5 ' — > 3 ' + Template-primer:

Primed single strands + + + •+ +

Nicked duplex ' + -• - -

Gapped duplex + + +

Polymer synthesis de novo +

-

Ribonucleotide incorporation

with Mn2+ +

Inhibition by -SH blocking + +

agents

Effect of sait (200mM NaCl

compared to 50mM, set at 100) 60 3 400

Size, daltons 109.000 114.000 80.000 122.000

Single polypeptide chain yes yes yes yes

Turnover number (relative to pol I) 2

Genetic locus pol A gene 4 3 gene 5

Phenotype of mutant, dna yes yes yes yes

Homogeneity of purified enzyme yes yes no no no Number of molécules/cell (est.) 400 600

(from"DNA synthesis" by A. Kornberg - Freeman Ed. San Francisco)

T4 DNA polymerase does not display 5' 3* exonuclease activity but does have an extraordinarily active 3'-> 5' exonuclease analogous to the "proof reading activity" found in E.coli Pol I (see paragraph on fidelity mechanisms) The isolation of a DNA polymerase from two specles of Mycoplasmatales has been recently reported (53), which does not possess any associated exonuclease activity.

The absence of the 3' -5» 5» exonuclease is particularly remarkable in that this activity is ubiquitous among the DNA polymerases that have thus far been

characterized from prokaryotes. (see paragraph on fidelity mechanisms).

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14.

Physiological rôles E.coll DNA Pol I

One of the most effective tools for assessing the rôle of an enzyme in vivo is the study of mutants affecting enzyme activity.

With this objective, several thousand strains of E.coli from a heavily mutagenlzed population were examined to find one, the 3478th, whose cell-free extract contained very low levels of DNA polymerase I activity

(53;a).This residual activity was later identified as a mixture of two distinct enzymes, called DNA polymerase II and III (40, 41) and poly- merase I was thought to be entirely absent. Since pol Al strain (mu-

tated for this enzyme) grows quite normally but is defective in its capacity to repair the DNA damage of ultraviolet irradiation and of radiomiraetic drugs like methylmethane sulfonate, Pol I enzyme was as- sumed to be essential during the repair but not in the replication of the E.coll chromosome (54). Recently however, with appropriate atten- tion to enzyme lability, extracts of ail pol A mutants were shown to contain measurable levels of DNA polymerase I activity and levels of 5 ' — ^ 3 ' exonuclease approaching that of wild type (55).

There are now évidences that replication of E.coli requires DNA polymerase I (56) and several proposais have been made for its func- tion in replication (57).

E.coli DNA Pol II

Conditionally lethal mutants in DNA replication are designated . as DNA mutants.

The known temperature-sensitive, ^na^^ mutants (dnaA, dnaB, dnaC, dnaE and dnaG) have normal levels of pol II activity. A mutant with no détectable (•<^0,5%) pol II activity in extracts, called pol B, has been obtained by assaying extracts from many surviving colonies of mutagenized pol Al cells (58). This pol A polB mutant grows normally at 25° and 42° and supports growth of a variety of phages. The pol B mutation does not make the cells sensitive to ultraviolet irradiation nor does it affect the frequency of recombination in transductions and mating transfers.

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Yet there are clear Indications that in mutants déficient in pol I and pol III, pol II serves in the repair of ultraviolet lésions (59).

E.coli DNA Pol III

The essentiel part pol III plays in DNA replication became clearer in studies of extracts from temperature-sensitive polC mutants.

The survey, done before pol Il-defective mutants were available, used phosphocellulose gel chromatography to separate pol III from

pol II. When isolated from dnaE, but not from other dna^-g mutants, the enzyme proved markedly temperature-labile (60).

Since DNA replication stops in dnaE mutants when the température is raised and since the pol III isolated from thèse strains also shows this température lability, we may conclude that pol III plays an essen- tiel part in DNA replication. The restricted template-primer capacity of the isolated enzyme (61) indicated that the enzyme functions in vivo with remarkably différent properties. Recently, during the investigation of the conversion of single-stranded circuler DNAs of the small phages

and ^y. 174, to the duplex circuler, replicative form, a more complex form of DNA polymerase III, nemed DNA polymerase III holoenzyme, hes been discovered (62)

T4 DNA polymerase

T4-induced polymerase was the first DNA polymerase directly corre- leted with chromosome replicetion. Mutents in gene 43 thet feil to induce end maintain significant levels of DNA polymerase also fail to maintein DNA synthesis even though related enzymes reach normal levels. (63)

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16.

Few notes on DNA replication

Thèse few notes will deal essentially with the mode of replication of bacterial DNA studied in vivo by a number of techniques and in vitro with the aid of simple viral DNA templates.

a) in vivo observation

The replicating chromosome has been observed in a variety of ways and in several organisms. Autogradiographic (64, 65) électron microscopy

(66) 67) and genetic (68) data suggest that DNA replication in E.coli and B.subtilis has a single origin and is bidirectional. Bidirectional replication also prevails in the more complex chromosomesof yeast (69) drosophila (70) and mammals (71) but apparently from différent initiation points forming several "replicQns" which are then fused together (72).

An interesting variant of the replication fork called "rolling circle" has been observed in many instances (73).

b) The replication fork

Because of the antiparallel orientation of strands in the double hélix DNA and since no DNA polymerase capable of polymerization in

3 ' - ^ 5 ' direction has been discovered, replication by the same enzyme cannot occur simultaneously and synchronously on both strands. This situation has been clarified when pulse-labelling with -^H thymidine have revealed 1000, -2000 nucleotide long "Oka^aki fragments" on the DNA at the growing fork, apparently synthetized in the 5' to 3' chain direction only (74) and subsequently joined together by DNA ligase (75)

(fig.3 part a and b) '

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polymerases to start DNA chains de novo (57). Such new chain starts are required at fréquent intervais on the lagging side of the fork (fig.3,a).

Partly for this reason, a new primer-generating polymerase (primase) was sought and eventually found in the E.coli repliçation System (76). Its properties suggest the possibility that Okazaki fragments are initiated in short RNA primers which are elongated by DNA polymerase^then cut-off and replaced by DNA before DNA ligase seals the lagging strand (fig.3 part b). The 5'-^ 3' exonuclease associated to DNA polymerase I could be a good candidate for the excision of RNA primers . (57)

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18

Other enzymes, like "unwinding enzyme" (77j ., helix-destabilizing enzyme (78) and the newly discovered "DNA girase" (79) are also thought to play a fundamental rôle in DNA replication.

c) in vitro observation

In vitro studies of the proteins required for the replication of E.coli chromosome have been undertaken with the isolation of condi- tionally lethal mutants - dna mutants - . They are defective in DNA replication in some restrictive environment, usually high température.

(80).

Cell free extracts (57) from thèse strains have been used to study in vitro replication (i.e. conversion from the single strand to the double strand form) of small bacteriophage DNAs as M^^' '^4»

^ X l 7 4 .

Complémentation assay (81)^using wild type cell free extracts as the donor of the enzyme activity complementing the déficient mu- tant cell free extract^allowed to the purification of the proteins required for DNA replication and have revealed that this process is exceedingly sophisticated : i.e. up to now eleven purified proteins have been shown to be necessary for the In vitro conversion of ^ X l 7 4 to the RF II form (81) ; T4 bacteriophage replication in vitro has been reported to require 6 purified proteins (82). Despite the fact that several proteins, of which counterparts have been proved to be necessary for DNA synthesis in bacteria, have been turned up in mam- malian cells (83)^attempts to détermine the rôle of thèse and other factois in eukaryotic DNA replication in vitro Systems have been

hampered by the apparent requirement for intact replication machinery and by the inaccessibllity of that machinery to manipulation in vivo.

However, some reports (83, 84) of in vitro studies on mammalian cells DNA synthesis are now available.

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Fidelity mechanisms

A most important feature of prokaryotic DNA polymerases as catalysts of polymerization of mononucleotides into long chains of DNA is that they can also dégrade thèse chains.

The pure enzymes can dégrade DNA from the primer t erminus in a 3 ' — ^ 5 ' direction ; this DNA polymerase-associated exonuclease has been shown to be capable to excise incorrect nucleotide incorporated into the DNA during the replication (85, 86) and thus termed "proof- reading exonuclease" (87) . .

In both E.coll (88, 89) and B.subtilis (90) some mutations that render DNA polymerase III température sensitive also increase sponta- neous mutation rates. It has also been reported that two température sensitive gene 43 mutants of T4 which had altered DNA polymerase also had strong mutator activity (91, 92).

A reduced fidelity of the T4 polymerase altered by the ts L56 mutation in gene 43 has been dempnstrated in an in vitro DNA polymerase System (93). Furthermore, purified T4 DNA polymerases from ts L56 mu- tants (mutator) and ts L141 mutants (antimutator) showed an altered ratio of polymerase tD3*-7>5' exonuclease activity, which was found in- creased in the case of the mutator and decreased the case of the anti- mutator polymerase when compared to the wild type enzyme (86, 94).

However an apparent exception is the mutator ts L88, which has nearly normal exonuclease activity but low polymerase activity at ele- vated température where polymerase but not the exonuclease activity is inhibited. However ts L88 enzyme displays mutator activity rather than the antimutator activlty expected if only the ratio of the two activi- ties was crucial to àccuracy (95). In addition to this, other results

(96) suggest that base sélection : i.e. discrimination against incorpo- ration of the incorrect ba«e (before the removal by the "proof reading' exonuclease) is also essential in maintaining the high fidelity of DNA synthesis.

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20.

"Kinetic proof reading" is an hypothesized feature of polyraerizing enzymes in which errors during protein or nucleic acid synthesis are reduced by the introduction of branched pathways and/or of a time delay, between the formation of an activated complex and the forma- tion of the product, during which incorrect aminoacids or nucleotides could be "kinetically" proof read and removed. (97, 98).

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EUKARYOTIC DNA POLYMERASES •

General properties

Higher eukaryotic cells contain at least three distinct DNA polymerases which have been named DNA polymerasés ts( , jl and (99, 100,

101).

Mitochondria isolated from mammalian cells also contain a DNA polymerase activity that has been considered to be différent from

othèr cellular DNA polymerases. However, récent évidence has suggested that the mitochondria-associated DNA polymerase may be a form of DNA polymerase^ (102, 103, 104).

Table III shows some properties of animal DNA polymerases.

It is interesting to note that a multiplicity of molecular forms having the same fundamental properties as the of -enzyme, but différent molecular weight^have been observed. While some heterogeneity may be

ascribed to aggregation artefacts in low ionic strength, the évidence for the permanent association of a simpler, catalytically active "core"

to other proteins seems convincingly demonstrated (105, 106).

• This brief introduction has been centered on work done with the mammalian DNA polymerases. Studies on DNA polymerases from other eukaryotes have been extensively reviewed. (107, 108 )

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T A B L E III

PROPERTIES OF ANIMAL DNA POLYMERASES <V , ^ and )^

Properties

Physical and chemical properties Molecular weight : native

Molecular weight : denatured (SDS) S value

Homogeneity

Isoelectric point

Tendency to aggregation

Chromatographic behaviour (order of elution with phosphate buffer, pH 7.5)

DEAE-cellulose Phosphocellulose Hydroxylapatite Native DNA

Functionaland catalytical properties In vivo localization

% of total activity growing cells resting cells Spécifieactivity (Units/mg)

Number of molécules/cell (estimated) Polymerization rate (nucleotide x molé- cule-'! X sec~^) (estimated)

Effect of DNA extending proteins Effect of histones

(f^, ^232' "^3 t^^sl histones) dNTP dégradation

Pyrophosphorolysis Pyrophosphate exchange

130-180,000 70-90,000

6 - 8 yes 5.6-6

yes

45,000 45,000

3.5 yes

9-9.4 yes

III

1,11"=

1,11'=

nucleus

> 85 0 -5 800,000

6x10"^

III

nucleus 10-15 90-95 200,000

7x10^^

>140,000^

7 - 9^

no 5.4-6.1

yes

II III III II

nucleus mitochondria

2-5 5-10 25,000^

30 2.5

stimulation no effect no effect inhibition

no yes yes

inhibition inhibition^

no no

no no

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i

Associated DNAses 3' 5*

5' •:> 3»

Associated RNAse H

Inhibition by antibodies against Inhibition by antibodies against (3 Inhibition by antibodies against Inhibition by antibodies against Reverse Transcriptase

Ability to copy DNA across pyrimidine dimers Km for dNTP»s (yWM)

Optimal pH

Preferred divalent ion : activated DNA synthetic polymers Hlgh ionlc strength effect

Effect of pCMB Effect of N.E.M.

Présence of Zn++ in molécule Template : primer complexes a) Homopolymers

deoxytemplate : deoxyprimer deoxytemplate : riboprimer ribotemplate : deoxyprimer b) Natural DNA

Activated native double-stranded gapped DNA

Nicked native double-stranded DNA (T5 phage DNA)

Denatured' Native DNA

RNA - primed DNA

no no no yes no no no

yes 2-12 7.2^

Mg++

Mn + + inhibition inhibition inhibition

yes

yes yes no

yes no no no yes

no no no no yes no no yes 8-12 8.5-9

Mg++

Mn^"*"

no no no yes no yes 0.2-0.6

8

Mg + + Mn + + . stimulation^ stimulation*^

inhibition inhibition no effect inhibition

yes

yes poor

yes

yes no no no no

yes

yes poor yes

yes no no no no Footnotes to Table III

a. Exception : liver mitochondrial polymerase

b. Not adsorbed at 0.02 M K-phosphate buffer, pH 7.5 c. Poor séparation

d. Best partial purification ( 107a)

e. Activated DNA was used as primer-template f. K-phosphate, 20 mM, is the preferred buffer

g. Maximal stimulation ih 50 mM Tris HCl buffer (pH 8.5) is obtained at 0.1-0.13MKC1 h. For combination of différent KCl and K-phosphate concentration see Réf. 107a

Highest enzyme activity is obtained in the présence of 50 mM K-phosphate and 100 to 200 mM KCl

From "The three DNA polymerases of animal cells ,; properties and functions" by A.Falaschi and S. Spadari - in "DNA synthesis présent and future" Plénum Press London.

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24.

Physiologlcal rôles DNA polymerase P(

The lack of conditional mutants makes difficult to ascribe un ambiguously a biological function to a given enzyme ; however several U n e s of évidence suggest that DNA polymerase «< is responsible for DNA replication in Eukaryotic cells :

a) Its polymerizlng rate per molécule is faster than that of the

(3 -enzyme and is close to the velQcity of the growing chain in vivo ( 107b) ;

b) It is the only polymerase of which level constantly correlates with the DNA replication rate (109, 110) ;

c) It is the only polymerase that can use the RNA-primed natural DNA as template (111) ;

d) It is the only polymerase stimulated by the "DNA extending proteins"

(112)

DNA polymerase ^

The poor or nil corrélation of the level of this enzyme with DNA replication rate, and its constance in différent physiologlcal conditions has invited the suggestion that it may be related to repair type syn-

thesis. A more direct indication in this sensé cornes from the work with the Phytohemagglutlnine stimulated lymphocytes : thèse, besides the in- crease of o( -polymerase in correspondence to the peak in DNA synthesis rate, show a second increase of DNA polymerase activity (113) occurring at later times, when DNA replication rate is at a minimum. This second wave of DNA polymerase is parallel to a peak in the level of other en-

zymes acting on DNA, like DNA ligase and a DNase acting on single stranded DNA (113). The second polymerase peak is due mainly to the polymerase|3, which reaches only at this late time (2 or 3 days after the peak in DNA synthesis rate) its maximum level in this system, and is indeed the préva- lent polymerase in the lymphocytes at this stage (114). This late peak of DNA enzymes corresponds to a peak in the capacity of the stimulated lymphocytes to perform repair synthesis following UV irradiation.

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DNA polymerase |^

The location of this enzyme, in part at least, in the mitochondria (102) where it is the only polymerase présent, strongly suggests that one of its rôles is the replication and/or the repair of mitochondrial DNA. An involvement in repair synthesis may be suggested by the obser- vation of the low Km, whereas a main rôle in replication of nuclear DNA seems unlikely from its scarce response to proliferative stimuli and its low polymerization capacity.

Fidelity mechanisms

As more eukaryotic DNA polymerases were increasingly purified, it became apparent that the nuclease associated activities found as intégral part of the prokaryotic DNA polymerases (see table I, this introduction) were not characteristic of thèse enzymes. No eukaryotic DNA polymerases have been reported to have 5'-^ 3' exonuclease activity

(114a, b,c -) ; highly purified«-polymerase (from KB cells (114d) sea urchin nuclei ( 114e) and calf thymus ( 114a) showed a reduced, but still détectable, amount of 3'—>>5' exonuclease activity while^ poly- merase from calf thymus showed no exonuclease capacity at ail (ll4c).

Thus, if incorrectly base paired nucleotides are incorporated into DNA they may be excised by other enzymes in eukaryotic cells (see Article 3 ).

Recently, however, the purification of a new mammalian DNA poly- merase termed polymerase ^ has been reported ; this polymerase seems

to be associated with a very active 3 » — ^ 5 * exonuclease (114f).

Despite the virtual absence of 3'-^ 5' exonuclease, some eukaryotic DNA polymerases have been reported to be extremely accurate in copying homopolymer substrates in vitro (114g,114h) sùggesting that at least in thèse in vitro Systems proofreading is achieved at a step prior or at the moment of incorporation itself, maybe via a base-selection mecha- nism similar to that postulated for prokaryotic enzymes (see Fidelity mechanisms in prokaryotes, introduction I).

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26.

RNA-dependent DNA polymerases

The finding that a DNA polymerase in the virions of RNA tumor viruses, which could use viral RNA as a template to direct the incor- poration of deoxyribonucleoside triphosphates^ was an important event.

The discovery of the so-called reverse transcriptase (114i,n41).

supports the notion that RNA tumor viruses replicate via a DNA inter- mediate of which genetic information can be integrated into host chromo- somes (114m 114n).

RNA-dependent DNA polymerase has been isolated from virions of both avian and murine RNA tumor viruses.

The enzyme•isolated from the avian myeloblastosis virus (AMV reverse transcriptase) has been purified to apparent homogeneity (115) and showed to incorporate an exceptionally large number of incorrectly paired bases copying a variety of ribonucleotide and deoxyribonucleotide templates (116, 117). The most purified préparation of AMV reverse trans- criptase has been shown to be free of any associated nuclease (115).

Terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is an enzyme that Catalyses the elongation of preformed oligomeric or polymeric DNA chains by adding deoxyribonucleoside monophosphates to the 3'OH ends of thèse chains (118).

Unlike the other DNA polymerases, this enzyme does not use nucleic acid template for instruction, but acts by terminal addition to single chains Terminal deoxynucleotidyl transferase is found in large quantities in thymus (117) and is believed to be spécifie for thymic or prethymic cells recently the enzyme has also been found in large amounts in circulating lymphocytes of patients of acute lymphoblastic leukemias (119) and some other leukemias (120, 121).

The function of the enzyme is unknown, although a rôle in im- munological diversity has been proposed (122) ; however its capacity to polymerise independently from a template instruction makes it an idéal candidate for the rôle of an "error pronepolymerase" (123).

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- EXPERIMENTAL STRATEGY AND GOALS OF THIS WORK -

In vivo studies performed both on bacteria and mammalian cells indicate that ultraviolet induced lésions in the DNA cannot be copied by the normal DNA replication machinery (see Introduction I)

Detailed genetic évidence indicates the existence in bacteria of an inducible mutagenic pathway of DNA repair which has been termed "SOS System" and the occurrence of which has now been postulated also in mammalian cells (see Introduction I).

In vivo biochemical data obtained on the replication of UV-irra- diated bacteriophage 174 DNA suggest that the mutagenic character of the "SOS System" could arise from the induction of an "error prone"

DNA synthesis capable to polymerize through the lésions in the bacterio- phage DNA. We have undertaken an in vitro study on the rôle played by DNA polymerases in the replication of damaged templates in the attempt to evidentiate the induction either of a new "error prone" DNA polymerase or of a factor(s) affecting the fidelity mechanism of the constitutive polymerases.

A major fidelity mechanism, 3' to 5' exonuclease (proof reading), has also been studied as the capacity of DNA polymerases to excise

3' OH terminally mismatched bases during in vitro polymerization.

Furthermore a putative "proof-reading" 3«-^ 5' exonuclease has been isolated from mammalian cells and its capacity to affect extent and fidelity of the in vitro DNA synthesis by the o( polymerase on intact and UV-irradiated templates has been investigated. We have also examined the effect of a mutagenic and carcinogenic métal (Be''"'") on the polymerase nuclease complex.

Thèse in vitro studies were undertaken in the hope to contribute to the elucidation of the molecular mechanisms involved in induced muta—

genesis in both bacteria and mammalian cells.

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28.

- EXPERIMENTAL SYSTEM -

- Cholce of template-prlmers for In vitro DNA synthesis

a) j> K 174 template-primer complex was constructed using single stranded phage DNA to which short complementary fragments of X 174 RFi DNA (covalently closed, replicative form) were annealed. ( see Article 1

and Article 3 )

b) Polypyrimidine homopolymers (poly dC and poly dT) , primed by short oligomers of complementary bases, have been used as a tool for

measuring "DNA polymerase infidelity" in vitro ; the error fréquency was estimated as the ratio of non-complementary to complementary nucleotide incorporated. (see Articles 2 y 3 and 4)

A simplified scheme of this reaction is shown below.

dAP... dAP,..

dTP... dTP... dTP... dTP... dTP, dTP poly dT;oligo dA

correct synthesis

dAP-dAP -dftP_dfiP _dftP_df)P dTP-dTP-dTP-dTP-dTP-dTP

Mg^^ (or Mn**) Enzyme

\ d A P - dAP- clftpjcicp|. (iRP-ûlR P dTP-dTP-dTP-dTP-dTP-dTP error rate 1 in 4

Polypyrimidine homopolymer templates were used in order to obtain large amounts of pyrimidlne dimers following UV irradiation, which was used as the source of mutagenic DNA lésions.

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;) An homopolymer carrying at the 3«OH terminally mismatched bases (see below) has been utilized to investigate the 3 » — ^ 5 ' exonuclease

"proof reading" activity of the DNA polymerases (Article 3).

dC-dC-dC dT-dT-dT-dT'^

dA-dA-dA-dA-dA-dA-dA-dA-dA-dA Choice of the sources of enzymatic activities

a) bacteria : tif-1 induced bacteria (either pol^ and pol A strains) have been preferred to UV-induced bacteria as source of DNA poly- merase activities because they can be handled with much more ease, in particular for large préparations ; furthermore, they gave a lower background when used for misincorporation experiments (see Article 2). E coli wild type and pol A strains, which do not carry the tif-1 mutation, have also been used as source of DNA polymerases

(see Article 1).

3

b) mammalian cells : ^ -irradiated lOT-1/2 CH mouse embryo cells (Appendix IV) hela cells (Article 3) and calf spleen (Article 3) have been used as sources of DNA polymerase and nuclease activities.

The particular advantages of using calf spleen as source of purified enzymes for the purposes of this work are the following :

1) over cultured cells : absence of common and troublesome contami- nants of cell cultures as mycoplasmatales (see Introduction II) ;

2) over calf thymus : absence of deoxyribonucleotidyl transferase activity (see Introduction II).

Purified avian myeloblastic RNA dépend DNA polymerase (see Introduc- tion II) has also been used (Article 1).

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30.

R E S U L T S

Article 1 : Mechanism of ultraviolet-induced mutagenesis:

Extent and fidelity of in vitro DNA synthesis on irradiated templates.

Article 2 : The molecular mechanism of induced mutations and in vitro biochemical assay for mutagenesis

Appendix I : Evidence for an induced error prone DNA synthesis in E.coli cell-free extracts on poly (dC): oligo (dc) template-primer.

Appendix II : Attempts to purify an "error prone DNA polymerase"

activity from SOS-induced E.coli mutants

Appendix III : Evidence for the induced inhibition of a 3'--^ 5' exonuclease activity possibly associated with DNA polymerase III in a SOS-induced E.coli mutant.

Appendix IV : Attempts to evidentiate the induction of an "error prone"

DNA polymerase activity in X irradiated mice cells.

Article 3 : Possible involvement of a "proof-reading" exonuclease in mutagenesis of mammalian cells : biochemical studies

Appendix V Purification and partial characterization of a 3'-^ 5' exonuclease from calf spleen.

Article 4 : On the mechanism of Béryllium induced infidelity on mammalian DNA polymerases.

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ARTICLE N° 1

Classification : Biochemlstry

Mechanism of ultraviolet-induced mutagenesis :

Extent and fidelity of in vitro DNA synthesis on irradiated templates • (pyrimidine dimers/DNA polymerases/proof-reading exonuclease/misincorporation)

GIUSEPPE VILLANI, SERGE BOITEUX and MIROSLAV RADMAN Département de Biologie Moléculaire.

Université Libre de Bruxelles B 1640 Rhode St Genèse Belgium

Proc.Natl. Acad. Sci. U.S. July (1938)

Abbreviations : UV, ultraviolet light; Py-Py, pyrimidine d'imer;

AMV, avian myeloblastosis virus.

• This is the second paper in the séries "Molecular Mechanisms of Induced Mutagenesis" (6)

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32

ABSTRACT

The effect of ultraviolut IrratJiatiud OD the exltMit and Videlity of

DNA synthGsis in vitro was btuLiiod usiny priiiiijd sini'.lu uLranded 0X17-1 |Jh:ii?i ;)MA and honiopo lymers as aubatrates. UnFract Joikitod and Trai:! inriatiid cell-froG t.-\iiai:L

+

from E.coli pol and polAI mutant bacteria as W G I I as purifiad DNA polynuîrase I (deoxynucleosidetriphoaphate : DNA dGoxynucleotidy1transferase EC 2.7.7.7) werG used as sources of en2yinatic activity. The oxtent of inhibition of DNA synthesis on ultraviolet-irradiatod 0X174 DNA suggested that pyl'imidiiit; diiiiers act a:i .JH absolute blocK for.chain elongation by DNA polyrtiorases î and III. Experimonts with irradiated pQly(dC) templato failod to detect incorpoi aior

of noncomplementary tjases due to pyr.iiiiiLjine tliinei s. A large inci'Gase in ihu turnover of nuclooside ti'i(Jhos|Jhates to freu inonophosphaciis dui^ing syntho-.ii:; by DNA polyrnerase I on irradiated 0X174 DNA has been obsorved. We propose that. tliis nucleotidG turnovGi' is duo to idling by DNA (iolyiiun\iS£Jl i . o. j ncorporu t ;i on anu' subséquent excision of nucleotides opposilio UV-photo 1 osions, by tho .V-» 'J '

"proof-road'lng" oxunucl.jriLjL:) thus prévint i ng repl :ii:at ion pa;it

pyrirnidine diiiiers, and the potentially mutagenic tîvunt t.bat shuuid lusuJt..

In suppoi't of this hypottiosis, DNA synthesis l.jy DNA ()o i yinorLiso li'Oin avian myeloblastosis vii'us (DNA nuclGotidyltransferase ; deûxynucleositJetrlphospli.u.iJ

ÛNA deoxynucleot i dyJ transf uraso ; ED. 2.7.7.7.), and l.y m ïiimial ian DNA pal yinor. iso a bûth of whlch are devuiti o I'any exunuoleaso ai:tivity, UMS fouiid tu bo on 1 v nar r i a ) 1 y

inhibited but not blocKed by UV irradiation ol' the toinpiato, and acconipanioLl by an increased ' incorporation of noncomplementary nucleutidris. It is

suggested that ultraviolet mutagenesis in bacteria roiiuii-os an inducod

modification of the cellulai- DNA replication iiiachinory, (lussibly an intvibitiuji of the 3' -»• 5 ' exonucleasG activity associated with iJNA po 1 ymx.'rases.

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UV irradiation of \ and 0X174 bactfsriaphagoa rasults in KilJina, but no

mutagenesis, of phago following infection op untreated hast cells (1 - 2), whareati.

UV irradiation of the host cells causes mutagenesis of botli untreated and irradiateu phage- (1 - 2 - 3 - 4]. Furthernioi-e, UV-irradiated single strancJed 0X174 ÛNA

extracted from untreated host cells was found to be replicated, only to the

first pyrimidine diiiier (5 - 6). Cai 1 let-Fauquet et al. 16] found that UV irradlatioi, of the host cell leads tq an epihancement of the DMA syrittiesis on UV irradiatcJ

0X174 DNA in vivo, which led them to propose that pyrimidine diniers in the 0X174' DNA can become mutagenic through misincorporation of deoxyribonuc1eotides

opposite pyrimidine dimera.

This inducible mutagenic system is thought to be pai't of a complex cellular emergency response ("SOS induction") triggered by unrepaired DNA lésions (sLjch as pyrimidine dimers), which also includes the arrest of cellular division, the

arrest of aérobic metabolisni and prophage induction in lysogenic bacteria (7 - ii ) . DNA polymerases are Known to be able to contrai the Fidelity of DiJA synchusiij by sélection of the correct nucleotide (9) and by possession ol" a "pi oo F-reai.llng"

3'-+ 5' exonuclease activity that can detect and excise 3' terminal iiii siiiatcheil bases (10 - 11 ] . , • .

We have sought to understand the molecular mectianisuis unriei lying UV mutagenesis • tiy an examination of the rôle of DNA polymtirases in the in vitro replication uf damaged DNA templates.

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34.

MATERIAL AND METHODS

Bacterial and P\:\aç]e Strains. E.coli HF suïl"*" ami HF A7m , thyA~ su"

were used as permissix/e and non-porinissivu host -strains for 0X17''l Eani 3 pliage, E.coli GC 714 (his-4 praA2, arqE3, lac"^ galK2, str-3, sFiA11 tif-1 . uvrA]

E.coli DM 1180 (Thr leu, his ilv^ str tif-1, sfiAII, lexA3j and

>. , |3S .— .—

E.coli HMS'SO (PO1A1 endoT Thy ) were prouided by Drs. J. George, D. Wount and C. Richardson, respectiwely,

Enzym&s, Hornopolyiners and other' Materials. E.coli DMA polymer'ase III was fraction \J of purification scheme of Livingstone and Richai''dsQn (24), DNA polymerase I "large fragment" according ta Setlow et al. ( M ) , was

purchased from Boeringher, Mannheim. One unit of enzyme activ/ity incorporâtes 10 nmoles dNMP into acid-insoluble niaterial during 30 minutes at 37°C under the reaction conditions of Richardson (is). DMA polymerase u from calf spleen was purified through DEAE cellulose, phosphocellulose, hydroxylapatite, and DNA cellulose column chromotography (31). . •, '

E.coli DNA polymerase I and AMV DNA polymerase ("Rev/erse transcriptase"]

purified to apparent homogeneity (l6], were kind gifts from. Dr. L. Loeb.

Photoreactivating enzyme purified from Streptomices griseus was a gift of Dr. G. Veldhuisen, University of Leyden.

Blu II restriction enzyme, which is an isoschizomer of Hae III restriction enzyme (l7), was prov/ided by Dr. M. Van Montagu. • .

Poly(dC) and o l i g o ( d G } w e r e from Collaborative Research.

14-

C labeled pQly(dT) and oligo(dA) were kindly prepared by Dr. F. Campagnari.

Unlabeled deoxynucleotide triphosphates were purchased from Boeringher.

3 32 14 3 H and P labeled deoxynucleotides triphosphates and C and H Thymidine were purchased fi^om Amersham Radiochemical Conter (u.K.).

Polyethyleneimine-impregnated cellulose thin loyer plates (iLC plastic stieets PEI-cellulose F) were from Merck,

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Prlined 0X174 LINA .

' Template-priiiitiP coinplex waa constructei] usiiig l''*--] labelud sinp.lt:' str.iiiaeiJ ptiage 0X174 DNA to which LiharL coinpleiiientary l'iMciiuJiil-i:; uf 0X17'1 RF-I flNA

(covalently closud, repJicative fonn) woru aniu.'rjjLid. fi-ep^irat iun oF tnu ivto DNA speciûs lias liuen dcscribed [101. Tlie r.pGcinc rcidioact: 1 vity was 4.11) L:piii per single strand molécule. Purified I^F-I supci-coiled IINA was nlcKed eil.her liy pancreatic DNAse [method A] or Blu II restrictian enzyme (method D] (détails wlll be published elsewhere). Hethod^A : NlcKed RF-I DNA molécules were mlxeJ in eciual nucleotide amount to [ ' V ' ] labeled single strarided DNA (final concentration

5^(pyml) into 10 riiFl Iris pli U, 10 md EDTA and 10 niM N.iCl . DNA was denaturatoLl by addition of final 0.1 N NaOH at 42° for 10 minufes. Tlie mixture v-var, neutr.j lized by a 10-fold dilution oF l.fl M Tris IICI, 0.2 M li-Ls Base pli G.I.

Annealing of homologous DNA fragments was achieved by addition of 50

formamide (MercK) for' 2 haurs at room temperatui"e and lollowod liy dialysls against 50 mn Tris pli B, 1 inN EDTA and 10 mM IMaCl. The resulting DNA was

.characterized and puriFied by sédimentation through neutr>il sucroise s'.raifiont. /\

sédimentation coeFFicient S.,,_, ,, 24.0 corresponds ta 0X174 single sLrandiMl molécules which are in average 15 % double stranded. Visualisation oP ttiis DNA tay électron minroscopy, Kindly |)crfoi'med by Dr. 0. rii.chel -Maenhaut, has shown circular ptiag^e 0X174 DNA mostly with one double-strandeLl Fragment.

[*lBthod_B : 5D^,ug/ml 0X RF-I DNA was cleaved' to saturation by Blu II restrùjt ian endonuclease in 10 mM Ti'is pli 7.5, (3.6 mN n g C l ^ , G ml4 /9-merciDptoethancjl, Fur 4 hours at 37°. Reaction was stopped by adtJition of FDIA to a Final concentra- tion of 150 mM. Ail DNA was cleaved into 11 fragments, as observed in 1.5 l

agarosG gel electropiioresis, inixed in a 1:3 ratio to | C.j labeled sinj'.le si.randi:

0X174 DNA and annealed as described in method A.

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36.

Ultraviolet irradiation of primed templatos. UV irradiation af honiopolyiners (in 50 mM Tris pH B, 2Û mM KCl) and of ^"^C labeled primed ^X174 DNA

(in 50 mM Tris phi 0, 1 mM EDTA, 10 niM NaCl) was carried as described • (e).

Fluences were measured by a Latarjet dosimeter.

In vitro pliotoreversal of pyrimiriine dimers. Photoreactivation of UV irraciiated 0X174 DNA was carried out in 0.15 M NaCl, 0.015 M sodium citrate pH 7.0 for

1 hour by flash photolysis at 37° in the présence of saturating amounts of

photoreactivating enzyme. The source of flash light was at 10 KW-sec filtered through glass and water layer.

Cell free extracts. Cells were grown ta a concentration of 5.10 bactei-ia/ml in rich médium (10 g bactotryptone, 5 g yeast extract, 10 g NaCl per liter) supplemented witfi 15 t^g/ml of thymidine when required, centrifuged at room température, far 10 minutes at 8000 xg a n d r e s u s p e n d G d in 0,1112 v o l u m e of

10 % sucrose, 50 mM Tr'is HCl pH 7.5 at room température, quickly fi^ozen in liquid nitrogen and stored at -20°.C until use. Cell-free extrac'ts were prepared as described (l9). .

Assays for DNA synthesis. The assay mixture (O.l ml) for E.coli pol"^ cell free extract and purified DNA polymerase I cantained 50 niM Tris HCl pH B,.

6 mM MgCl^i 1 dithiothreitol, 2 nmales of each of the four deaxyribonucle- 14

Gside triphosphates, 100-200 picomoles nucleotide o f primed C labeled

ÇÏX174 DNA and either 50-100 jig proteins of cell free extract or 0.07 units of DNA polymerase 1. The assay mixture (o.l ml) for, E.coli polAI cell free extract was the same as. far purified DNA polymerase III and cantained 50 mM Tris HCl pH 7.4, 10 mM MgCl^, 5 mM dithiothreitol, 5 mM ATP, 5 inM spermidine HCl,

5 nmales each of the four deoxyribonucleoside triphosphates, 400-500 picomoles 1 4 '

nucleotide of primed C labeled ^/jX174 DNA and 5(3-100 ^g proteins of cell 3

free extract. The H labeled deoxyribanucleoside triphosphate had a spécifie activity of 500—1000 cpm/picomole. Reuctians were carried e u t at 30°, âliquots were withdrawn a t indicated times and spotted anta CP/C glass fiber filters., washed by the method af Dullum {','\>] and theii" l'adioactivity was measurtJd in • Omnifluor scintillation fluid.

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Assays for misincorporatlon, are described in the legend to Table II.

Assay For incorporation and hydrolysis of newly incorporated nucleotides. Both incorporation and hydrolysis of newly incorporated nucleotide monophosphates were measured using one-dimensional thin layer chroaiatography to separate radioactive DNA tcmplate, labeled dNTP and the dNMP derived from labeled

dNTP, when the reaction was completed. Polyethyleneiniine thin layer chroniatograph- was performed as described by Hershfield (32) , except that the areas correspondiri;

to dNMP, dNTP and DNA template (the origin) were scraped off and their radio- activity measured directly in Qninifluor scintillation liquid. The substrate

1 ^ 3 '

was primed' C labeled (/^X174 DNA and the labeled nucleotides were H dlTP or dATP.

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RESULTS ^ . .

Inhibition aï' Jn '^fû ^NA SyntfiesiB by E.coli DNA' Pùlyiiiarase I and 111 on UV-irradiated 0X174 DMA,, •, . '

Single stranded 0X174 phage DNA was primed by a n n u a l e d fragnientt; of 0X174 RF-I DNA as dt^scribed in Materials and Metliuds. Up to IDD p e r c u n t of the input non-irradiatod 0X174 DMA teinplate can be replicaLed in polyiiieriza- tion reactions by ceil free extracts from E.coli pol [F.ig.1a), po 1 Al (Fig.lb) or by purified E.coli DNA polyiiierase I "l-arge fragment" (pig. 1c].

In agreement with previous studies (12, 13, 23, 29], ail three polymer-ization réactions were siinilarly inhibited by UV-irradiât ion oF 0X174 DNA ternplate

(Fig.1a,b,c) . The dose response o f the Inhibition ot DNA syntliesis suggests that each pyrimidine dimer is both an absolute blocK to polynucleotide chaln elongation in vitro a n d a lethal hit in vivo (T:ible l ) , a s uav, pr'eviously s h o i - ; i ,

by in_ vivo studies (C). This' conclusion is S L j p p o r t e d by tlie finding that in vitro monomerisation op pyrimidine dimers by photui-Oc-jctivation enzyme i;jn restore approximately 2'j % of the t e m p l a t e activity [Fig.la]. The probable;'

r e a s o n that the r e s t c i r a t i o n o f t e m p l a t e activity w a a n o t inoi-e orficient i n t h . i

only one.of the four possible isomers of pyrimidine ilimers induL;ed by UV irradiation of the single stranded DNA, can be photor-eactivated ( 22i .

Turnover op Nucleoside Triphosphates during DNA synthesls Ijy DNA Polymerase 1 on DV-irradlated template^.

'•E.coli DNA polymerase I' and III possess tho "prooP-readl ng" 3' - k - ' j '

exonuclease activity which could alearly.be involved in the failure op thesu enzytnes to fepllcate a damaged tempJate. To examine this possili i 1 i t y, both incorporation of niicleaside inonophospliates from l a t i e l e d Iriphosphate;; into primed 0X174 DNA and production oP Pree nucleoside mùnoplio'.iph.ites l'rjjm labeled triphosphates were followcd in a^ single assay dur-ing synthesis by DNA polymerase I (large fragment] on unlrradiated and i r r a d l a t e d (INA (r.ee Materialsand Methods a i i d l.egond t o I ig.2].

(46)

This analyslii was Uono with purifiod DNA ( l o J y i n u i \ i a i 3 fraction that w a s freo of DNA-indopomJont trlphosphatastas. In a d d . i t l n n , tho larj'.o f r a p j i K i n t ot' DNA polymcrases I (1^], iiiisBini^ the G' 3 ' i;xont.ic.loa!>o tnicK l i a n G l a t i n n ) , but possessing tho 3' -> 5' e x o n u c l e a s e aotivity, V-AJS u i j u d to avoid po^iîilile

p r o d u c t i o n of free innnuphosphates by tho 5'- 3' exonuclaase deiy~adatlon of

n e w l y incorporated ntjcliîoaide ninnophosphatès. F'ioiiulta oUtainad with L'^tJtJTlP (Flg.2a) and ['VlJ tlA'IR [Fig.Zb] as precursors clearly shLJwud an incroasud,

c o n t i n u e d production of froe monophosphates evon afti.-r a complète arrest of DNA synthesis on UV-irradiatcd 0X174 DNA. ûn the cantrary, DNA synthesia on no'nirr^jdiàted DNA prodiiced only small ainoLints of frot; monophosphates.

This resuit indicates that the arrest of DNA synthesit. by p y r i m i d i n a diir,ars i'j

accompanied by an jncreasod turnover of nuclooaitJu l.riphus|)hiJLus. Whi;n total (lUoltiotido tui-nos/er was calculated from fiî',.2, nn proTiirence for dATP turnov.-r overthedTTP turnover was obseived with UV i r r u d l. i L o i l tenipiate. irio d i n a r a n c c s

observed using unirr'adiated te(n(]late-are not very accurate^ in view of the small amounts of free monophosphates produoad.

The observed p r o d u c t i o n of n u c l e o s i d e monophQS|")hLites by DNA pc51 yinei-ase 1 on a damaged template, even in tlie absence op detectcibJe' DNA syntliasis, could be 'explained by rei'jeated attempts of the i)olymer>.ise to incorporate

nucleotides opposite pyrimidine dimei', followeti liy e x o n u c leoly t ic ' 3 ' 5'

e x c i s i o n of the resulting n u c l e o s i d e monophosphates. We will refer to this phenomenon as p o l y m e r a s e "idli.np".

I

(47)

40.

Extent oF DNA synthesis on UW-lrradiateri i/\K\7^] DNA by AMV DNA polyiiierase.

IF the 3' 5' exonuclcase activity oF E.coli DNA polynierases I and III

removes nucleotides inserted opposite (or near) pyrimidine dimers, tliereby ' effectively blocking replication past dimers, then DNA polymerases lacking the 3'->5' exonucleasG may replicate a damaged ternplate more eFFiciently than polymerases having this activ/ity, Fig. 3 shows that this is true For AMV

reverse transcriptase, an errorvprone DNA polymerase devpid of any exonuclease activity ( l 6 ) , which copies UV-irradiated 0X174 DNA Far above the level

copied by E.coli DNA polymerase I. . . ..

Fidelity and Extent oF DNA Synthesis on UV Irradiated Poly(dCl Template.

Pyrimidine dimers are bulky structural deFormations in DNA and are the

principal UV-induced mutagenic lésions (s), which drastically aFFect hydrogen bonding propertiés oF the involved bases, IF they were Frequently copied, the overall eFFect would be a higher rate oF incorporation oF incorrect bases than on an unirradiated template, To study the Fidelity oF i_n vitro DNA

synthesis, three diFFerent DNA polymerases were tested with UV -irradiated and intact poly(dc) (Table II] and poly(dT) (results not shown) hamofjolyners as templates. Synthesis by E.coli DNA polymerase III showed the highest

Fidelity 'and the greatest sensitivity to inhibition by UV-irradiation, whereas synthesis by puriFied, exonuclease-Free, mammalian DNA polymerase ii. and At.lV reverse transcriptase was less inhibited by UV-irradiation and showed a high increase in misincorporation [latale II).

Figure

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