Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III

Gene, !11 (1992) 43-49 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

43

GENE 06226

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III (polC gene sequence; hydroxyphenylazouracil resistance; mutator phenotype)

Marjorie H. Barnes, Russell A. Hammond, ChristopherC. Kennedy, Susan L. Mack and Neal C. Brown Department of Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655 (U.S.A.) Received by R.E. Yasbin: 28 June 1991 Revised/Accepted: 12 September/13 September 1991 Received at publishers: 15 October 1991

SUMMARY

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase Ill (BsPolIII), a 162.4-kDa [ 1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolllI unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segmer~t displaying homology with the Exo domain of Escherichia coil DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the G r a m - organisms, E. coil and Salmonella typhimurium.

INTRODUCTION

B. subtilis DNA polymerase III (BsPollll) is a Poi-Exo required for the replicative synthesis of B. subtilis DNA (Cozzarelli and Low, 1973; Low etal., 1976). BsPolIIl displays two features which clearly distinguish it from other Correspondence to: Dr. N.C. Brown, Department of Pharmaco~%:y, University of Massachusetts Medical School, Worcester, MA ~J|655

(U.S.A.) Tel. (508)856-2151; Fax (508)856-5080. Abbreviations: aa, amino acid(s); ara, arabinosyl (9-//-o-ar:~t~.nofuranosyi); B., Bacillus; bp, base pair(s); BsPolIII, PolIll of B..r,~bzilis;dNTP, 2'-deoxyriboNTP; E., Escherichia; EcPollIi, Poilll of E. coil; Exo, 3'-5' exonuclease; HPUra 6-(p-hydroxyphenylhydrazino)uracd; IC~o, inhibitor concentration (see Table lI, footnote e); kb, kilobas~:s or 1000 bp; nt, nucleotide(s); NTP, nucleoside 5'-triphosphate; oligo, oligodeoxyribonucleotide(s); Pol, DNA polymerase;polC, gene encoding BsPolIII; RFLP, restriction-fragment length polymorphism; S., Salmonella; ts, temperature sensitive; wt, wild type.

DNA polymerases of known sequence. The first is the enzyme's unique primary structure. Excepting a short segment of homology with the Exo domain of E. coil Poll and other Pol-Exos (Sanjanwala and Ganesan, 1989; Bernad et al., 1989), BsPolIll displays significant homology (Hammond et al., 1991) with only one other class of DNA polymerase - the Exo-deficient, replication-specific Polllls of the Gram- bacteria, E. coil (Tomasiewicz and McHenry, 1987) and S. typhimurium (Lancy et al., 1989). At this writing, Gram + - and Gram - -specific PollIIs appear to he, at best, distant relatives on a unique branch of the growing family tree of sequenced DNA polymerases (Delarue et al., 1991). The second, and most distinctive feature of BsPollll (and other Gram + bacterial PolllIs for which BsPolllI is a model [Barnes and Brown, 1979]) is its sensitivity to the inhibitory dGTP analogs of the HPUra type (Brown et al., 1986). No other class of DNA polymerase, not even the distantly related Gram- Polills, reacts significantly with HPUra.

44 The long range goal of our research on BsPolllI is to elucidate the three-dimensional, molecular basis for its catalytic function(s) and for the unique vulnerability of its dNTP binding site to HPUra. Our fu'st step in pursuit of this goal was to complete the sequencing of the BsPolllI structural gene, polC, and engineer it for over-expression in E. coil (Hammond et al., 1991). The second step, which we describe below, has been to manipulate specific polC mutations to formally locate the respective Exo and Pol domains within the BsPollll primary structure.

RESULTS AND DISCUSSION

(a) Definition of the Exo domain The paradigm for the structure of the Exo s;te of PolExos is the Y-5' Exo site of E. coii Poll, a zite which recently has been characterized in precise, three-dimensional molecular detail (Beese and Steitz, 1991 ; Derbyshire eta!., 1991). The upper part of Fig. I displays a comparison of the primary structure of the Poll Exo domain and a region of BsPolllI encompassing aa 415-570 (of., Fig. 1, part B). The extent of the homology and the conservation of aa residues involved in Exo catalysis in Poll (Beese and Steitz, 1991) have strongly suggested that the domain represented by the 415-570 block of BsPollll contains its formal

(A)

E. coll B. subtil~

~'~3

2oli III

Exo site (Sanjanwala and Ganesan, 1989; Barnes et al., 1989; Bernad et al., 1989; Hammond etal., 1991). To determine if the 415-570 block, indeed, resides in the Exo domain of BsPollll, we posed the following two experimental questions: (0Can the Exo activity of BsPolIII, relative to its Pol activity, be selectively suppressed by mutational alteration of aa resident in the 415-570 block? (ii) When such an Exo-deficient enzyme replaces the PolIII of a wt B. subtilis host, does it depress the host's capacity to edit newly rel~licated DNA and, thus, confer upon the host a 'mutator' phe, l~.,/pe? The rationale for the latter question is based on the observation that the E~o activity of a replication-gpecific Pol-Exo typically serves an editing functbn, px'eofreading the.terminal sequence,of the growing primer ~tr~d a~td remo,,~ngnoncomplementary nt residues ~;ccas~onallymisincorporated by the enzyme's Pol function (Ec~o!s et al., 1983; Reha-Krantz et al., 1991). Selective depression of the Exo function of such an enzyme therefore frequently increases the incidence of random genomic mutations, imposing a 'mutator" phenotype on its host (Muzyczka et al., 1972).

(1) Selective suppression of BsPolll! Exo activity Our initial strategy involved attack of specific aa within the putative Exo block, exploiting oligo-directed, sitespecific mutagenesis of the relevant pdC region (of., Fig. 1).

345-KLEKAPVFAFDTETDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDOISRE-RALE'LLK

PLLE

415-LLEEETYV~~LSAVYDTIIELA-AVKVRGGEIIDKFEAFANPHRPLSATI~Ei,TG~T~DML6

IA DE-RALKVGQNLK--YDRGILANYGIEL-435 • , ,,o.: ...... -: . . . . . .

(B)

. . .4 9 4 - A G R Y A A E D A D V T L Q L H L K M W P D : ,,: : : ,.

,

DAPDvv

~-COOH

a2sJ 415

1

605

1437

i

(c)

i[ ~n

ClaI

5 1

5

1

2944

BglII BclI

II

3416

3965

~NslI

4a5

L1

HlndII

I

I ..... ,

4395

4861

Fig. I. Structure of BsPollll and its structural gene. (A) Comparison of the aa sequence of the Exo domain of E. cog Poll with that of the segment encompassing aa 415-570 of BsPollIl, with the major region of homology shown in bold type. The starred residue represents that mutated to produce the two mutants described in rows B and C of Table !; IA denotes the site of the mut IA mutation (G430E; cf., row F, Table I). (B) An abbreviated primary structure of BsPollll, displaying the putative Exo domain (shaded segment between aa 415-605). (C) Abbreviated restriction map of the polC regions of the g. subtilis genome; the polC sequence, which begins at nt 45 and ends at nt 4356, is demarcated by the dotted lines connecting maps C and B; the stars emphasize the BgllI-Bcil fragment which contains polC mutations 22-27 (Table II).

45

The specific segment we chose to attack was FDVETTG (bold type in Fig. 1A), the homolog of the respective Exo domains of E. coil Poll (Beese et al., 1991; Derbyshire et al., 1991) and of the replication-specific polymerases of bacteriophage T4 (Reha-Krantz etal., 1991) and ¢29 (Bernad et al., 1989). Our initial target was E 427, the putative equivalent of E 357 in E. coil Poll (of., Fig. 1). Adapting the mutations which had worked to selectively eradicate the Exo activity ofE. coli Poll, we generated two mutant forms of BsPolllI in which alanine (E427A; Exol)or glutamine (E427Q; Exo2) replaced glutamate at residue 427. We engineered both mutations for over-expression in our E. coil-based plasmid:poiC expression system (Hammond et al., 1991) and found that both mutant versions expressed fully soluble protein with a good yield. The results of the

Catalytic analysis of these products are summarized in rows B and C of Table I. Although Ala substitution at the equivalent residues of the model Pol-Exos orE. coil, JZI29, and T4 selectively inactivated their Exo functions, we found that the mutation E427A eradicated both Exo and Pol activities. The more 'conservative' substitution of E427 with its analogous amide (Exo2) was more productive. This mutation depressed the Pol activity to approx. 10~o of wt levels. Nevertheless, its effect was clearly Exo-selective, depressing the latter activity to less than 0.2~/o of wt.

(2) Selective inhibition of Exo activity and the development of a mutator host phenotype To address our second question regarding Exo action vs. mutator phenotype, we attempted to introduce the Exo2

TABLE I Effects of mutation within the putative Exo domain of Bacillus subtilis Pollll Enzyme form

Residue change" wt --. mutant

Mutant oligo vs. wt sequence ~ showing codon/restriction site change

In vitro specific activity relatire to wt~ Exo d

A wt

None

Not applicable

Pale

Pol/Exo

Mutator phenotype; colehies per l0 '~ cells ¢ Strep. SO4

Novobiocin

(5 mg/ml)

(5 pg/ml)

I

!

I



0

<0.01

-

N.DY

N.D.

> >50

N.F. h

N.F.

...Ale...

oligo: T T T GAC GTC GCG ACG ACA GGA B Exol

Glu4Z7~ Ala (E427AI

Nrul Glu Thr Thr Gly wt: 1315-TTT GAC G T T GAG A C G ACA GGA

424-Phe A s p V a l

...Gin...

oligo: GAC GTC CAG ACG A C T GGA TTG C Exo2

Glu4-'7-.GIn (E427Q)

.... BslXI--425-Asp Val Glu Thr Thr

Giy

<0.002

0.1

0.11

0.44

4

12000

1300

0.6

0.6

!

< I


0.4

0

-

< I


Leu

wt: 1318.GAC GTT GAG ACG ACA GGA T T G ...Ghl...

oligo: ACG ACA GAA TTG TCA GCT GTA D mut-lA

Gly43"-, Glu i

(G430E)

PvulI

428-Thr Thr G l y

Leu Ser

Ale

Val

wt: 1327-ACG ACA GGA T T G T C T GCT G T A ..,ASh...

oligo: GTG AAC A A C ACA GGC C T T AAA A A T E mut-IB

F mut-IC

Ser~'-'l~ Asn i

Stul

($621N)

619-Val Ash Ser Thr Gly Leu Lys Asn wt: 1900-GTG AAC AGC ACG GGA C T T AAA AAT

Ala f'*-'-, VaP

Not applicable

(A662V) See Fig. 1. b See Hammond et al. (1991). F r a c t i o n V, prepared as described by Barnes and Brown (1979). a Assayed by the method of Low et el. (1976), using single-stranded D N A ,

Assayed with activated calf thymus D N A as described by Barnes and Brown (1979). r Determined by plating on C;o agar plates (Brown et al., 1972) at the indicated drug concentration; colonies were developed by incubation at 30°C. N.D. - not determined; N.F. - not feasible - mutation incompatible with host viability; Strep., streptomycin. g Sequence derived from polC-specific genomic D N A cloned from B. sub|ills rout-l, str.~.:-q 2355 (Bazill and Gross, 1973).

46 version ofpoiC into wt B. subtilis by transformation. However, the mutant gene product was apparently incompatible with transformant viability, obviating further in vivo experimentation. To f'md an Exo-deficient PolIII form which was suitable for in vivo analysis of mutator phenotype, we exploited a lead provided from the sequencing of the polC gene of the B. subtilis mutant, mut-1. Mutant mut-1 is a ts DNA synthesis mutant isolated from a chemically mutagenized population of B. subtilis (Gross et al., 1968); it bears a mutated form of polC which specifies a mutator phenotype and an unstable PolIII (Bazill and Gross, 1973). Recently, Sanjanwala and Ganesan (1991) reported that mut-1 consists of two point mutations, G430E and $621N, which they designated, respectively, mut-IA and mut-lB. To determine how these mutations affected the Exc function of Polill, we sought to confirm their sequence and examine the catalytic impact of each on the product of its in vitro expression from an appropriately engineered form ofpolC. We first cloned the KpnI-Clal fragment ofpolC of B. subtilis rout-l, (nt 1-2944; codons 1-966; cf., sections B and C of Fig. 1), exploiting the approach which we developed previously to clone the corresponding wt KpnIClal fragment (Barnes et al., 1~89). Our sequence analysis of the fragment indicated the present of not two, but three point mutations, the structures of which are summarized in rows D - F of Table I. The two mutations residing in or near the 'Exo box', mut-IA and rout-1B, were identical to those reported by Sanjanwala and Ganesan (1991), while mut1C, the third mutation at aa residue 662, was novel. Separate transformation experiments with an engineered version of the wt KpnI-Clal segment containing only mut-lC (results not shown) indicated that mut-lC was responsible for the rout-l-specific ts phenotype and polymerase instability; mut-lC transformants did not express the mut-I associated mutator phenotype (cf., row F, Table l). To examine the effect ofmut-lA and mut-lB on the Exo activity of PolIII, we engineered the respective version of polC for in vitro expression, exploiting oligo-based mutagenesis. The elites (cf., structure in rows D and E of Table l) were designed not only to introduce the desired mutant codons, but also to create a novel restriction site to simplify cloning and to provide a convenient RFLP marker with which to follow the mutation in subsequent transformation experiments. Both of the engineered mut-lA and mut-lB forms ofpoIC expressed soluble proteins in a yield comparable to that ofwtpolC; the catalytic properties of the respective form are summarized in rows D and E of Table I. Mutant mut-lB clearly did not exert a selective effect on Exo activity; it reduced both the Exo- and Pol-specific catalytic activities by 40%, producing an enzyme with an Exo/Pol ratio closely approximating that of wt PolIII. Mutant mut-lA also reduced both Exo and Pol activities;

however, unlike mut-lB, it had a clearly selective inhibitory effect on Exo activity, generating an enzyme with an Exo/Pol ratio less than 25~o that of the wt protein. To examine the relationship of Exo action and mutation rate, we introduced the respective mut-lA and mut-lB versions of poiC into B. subtiiis, using competent B. subtilis 168 ts-6 as recipient (ts-6 is a complex mutation ofpolC codons 940-941 conferring a ts (45 ° C) host phenotype; cf., Hammond et al., 1991). Genomic DNAs of ts + transformants (i.e., transformants wt with respect to the ts-6 allele) were subjected to restriction endonuclease digestion with appropriate enzymes (PvuII for mut-lA suspects and StuI for mut-lB suspects; cf., elite structures in rows D and E of Table I). The digests were blotted and probed with radioactive wt Kpnl-CiaI fragment, using Southern-based methods described previously (Barnes et al., 1989). Transformants carrying the respective mut-lA- and mut-lBspecific RFLP were identified, and five of each class were compared with an equivalent isogenic wt ts + transformant for the presence of the mutator phenotype characteristic of B. subtilis rout-1 (Bazill and Gross, 1973). The assessment, which consisted simply of determining the incidence of colonies resistant to the two antibiotics originally used to assess the rout-1 phenotype, is summarized for typical transformants in the rightmost columns of Table I. The results indicated that the mutator phenotype is specifically associated with mut-lA - the mutation that selectively depresses PolllI Exo function; mut-lB had no demonstrable effect on the resistant colony development. All ofthe mut-lA transformants displayed an incidence of resistant colony development 103-104 times that of wt, while that displayed by mut-lB transformants was equivalent to wt.

(b) Delineation of the dNTP binding (Pol) domain by structural analysis of an established collection ofpolCmutations specifying HPUra resistance (1) Properties of the mutations The first significant clue to the location ofthe Pol domain of BsPolIII was the position of the azpl2 mutation at aa 1175 (Sl175A, Sanjanwala and Ganesan, 1989; Barnes et ai., 1989) which generates an enzyme resistant to the dGTP analog, HPUra (Clements et al., 1975). Although azpl2 reduces the enzyme's capacity to bind HPUra, it does not significantly alter its affinity for either dGTP or the other three natural dNTP substrates (Clements et al., 1975). The latter observation has suggested that aa 1175 per se may not be an integral part of the dNTP binding site, but merely a residue coincidentally residing near it in the native, folded protein. To investigate the latter possibility and more closely localize the Pol domain in the enzyme's primary structure, we sought to identify the position and structure of polC22, po1C25,polC26 and polC27, four other

47 spontaneous mutations ofpolC o f B . subtilis that had been selected on the basis o f H P U r a resistance (Gass and Cozzarelli, 1973). The effects of each o f the four polC mutations on host phenotype (data of Gass and Cozzarelli, 1973) and the behavior of the respective PoilII (our data) are summarized in columns A - C o f Table II. Three o f the mutations, polC25,polC26 and polC27 confer a ts host phenotype (cf., col. B); two of them (polC25 and polC27) confer a mutator phenotype (cf., col. B), a property which suggests a role o f the respective residue in d N T P binding and/or choice. One o f the ts mutations, poIC25, produces a polymerase that is inactive upon extraction - a property (Gass and Cozzarelli, 1973) which prevents conventional analysis o f its in vitro response to H P U r a and araCTP. Fortunately, neither polC25, nor any o f the other three poIC mutations significantly depresses PolIII-specific, ATP-dependent synthesis in toluene-permeabilized B. subtilis cells (Brown etal., 1972; Gass and Cozzarelli, 1973; cf., col. C of Table II). Using this NTP-accessible in situ system+ we confirmed and extended the findings o f inhibitor analysis reported earlier by the Cozzarelli group (Gass and Cozzarelli, 1973; Rashbaum and Cozzarelli, 1976). The results, which are summarized in column C o f Table II, indicated that the mutations not only confer the expected resistance to HPUra, but also variously affect the enzyme's capacity to deal with the d N T P sugar analog, araCTP. The polC27 increased the sensitivity o f the system to araCTP more than tenfold. ThepolC22 and polC26 reduced araCTP

sensitivity approximately threefold, whereas polC25 had a small positive effect on araCTP action, increasing sensitivity approximately 1.7-fold.

(2) Cloning and sequencing We mapped, cloned, and analyzed the structural basis of each mutation, exploiting an approach identical to that which we used previously to physically characterize the H P U r a resistance mutation, azpl2 (Barnes et al., 1989). We located each of the four polC mutations and its respective H P U r a resistance phenotype on a 549-bp BglII-Bcll fragment spanning nt positions 3416-3965 (cf., star~ed fragment on the abbreviated restriction map in section C of Fig. 1). Sequencing of both strands of the respective BglIIBcll fragments indicated that each mutant fragment contained a unique point mutation affecting a single codo~." as indicated in column D of Table If, the three ts mutatio~..~, po1C25, 26 end 27 were novel, whereaspoIC22, fortuitously, was identical to azpl2 [the sequence ofpoiC26 also was identical to that reported by S~,ajanwala and Ganesan (1991) subsequent to the submission o f this wo.rk~. The mutations were distributed in two 'pairs' (aa 1173/1177 and aa 1264/1273) separated on the primary structural map by 87 residues. (c) C o n c l u s i o n s and future d i r e c t i o n s

The major goal of this research was to approximate the location o f the major catalytic domains of BsPolIII as a prelude to a detailed, high resolution structural analysis o f

TABLE !I Properties ofpoiC22-27mutants A

B. subtilishosP

wt

polC22 polC25 polC26 polC27

C In situ Pollll activity~ in toluenetreated cells

B Phenotype b Plating efficiency

Mutation rate

37°C

b/s- ~ h/s ÷ x 109

1 1 1 1 1

51°C

! ! < 0.01 <0.01 <0.01

HPUra 37°C < 0.01 > 20 > 20 >20 >20

5 4 200 3 30

Specific d

ICsoc

D Mutation wt ~ mutant nt

aas (codon)

N.A. T--, G T--. G T--*Af T~C

N.A. Sert 175...Ala Leu117~_,Trp Vap2~3--*Asp Phel2~--,Ser

activity

13.1 5.9 9.7 8.5 5.9

HPUra

araCTP

0.7 125 ! 15 38 105

11 32 6 32 0.85

a B. subtilispolA59met his leu obtained from N. Cozzarelli (Gass and Cozzareili, 1973)was the wt strain. The four derivative isogenicstrains carrying po1C22-27were provided by D. Dubnau; each was selected as a spontaneous, HPUra-resistant mutant of wt at 30°C (Gass and Cozzarelli, 1973). b Data of Gass and Cozzarelli (1973). Log phase cells were prepared, treated with toluene and assayed for ATP- and PoIIl-dependent dNTP polymerizationas described by Brown et al. (1972). [3H]TI'P was used as the radioactive dNTP at a concentration of 10#M and a specificactivity of 2 #Ci/nmol. d Specificactivity is given in pmol [3H]TMP incorporated per l0s cells in 30 rain at 30°C. ICso is the inhibitor concentration resultingin 50% inhibition of ATP-dependentDNA synthesis in the presence of 10 #M competingdNTP (10 #M dGTP in the case of HPUra and 10 #M dCTP in the case of araCTP). f See also Sanjanwala and Ganesan (1991). g Superscripts refer to aa (codon) position in the PolIII sequence (Hammond et al., 1991) N.A., not applicable.

48 the enzyme's major catalytic sites. The pursuit ofthis objective has been successful. The association of Exo2 (E427Q) and the mut- 1-specific mutation, mut-lA, with the selective suppression of the enzyme's Exo activity in vitro and the mut-lA-specific induction of a mutator phenotype in vivo has, not surprisingly, formally located the Exo domain of BsPollII in a region whose primary structure is homologous to the Exo center of E. coil Poll. Our results also have extended Sanjanwala's and Ganesan's (1991) analys! ~Jf the complex mut-1 mutation ofpolC, and unequivocally defined a role of the Exo function of BsPolllI in in vivo editing. We currently are using site-specific mutagenesis and the structural information furnished by these results to further delineate residues within the Exo site that are directly involved in to Exo catalysis. The definition ofpo!C22-27, four mutations which variously and specifically affect the enzyme's capacity to bind two structurally distinct classes of inhibitory dNTP analogs, has localized a dNTP-reactive portion of the enzyme's Pol domain to a 98-aa segment (1175-1273) in its distal C-terminal fourth. The primary structure of this segment appears unique; it displays no striking conservation in comparable segments of the distantly related polymerase-specifie (00 subunit of Gram- bacterial Polllls (Tomasiewicz and McHenry, 1987; Lancy etal., 1989; Hammond etal., 1991). Nor does it display significant sequence homology with the putative dNTP binding sites of E. coli Poll and other members of the major DNA polymerase families (Delarue et al., 1991). Although our data strongly suggest that the aa residues at the ends of the 1175-1273 segment directly react with dNTP, they by no means reveal whether this segment constitutes the entire Pol and/or dNTP binding domain within the folded enzyme. To address the latter issue, we currently are exploiting random mutagenesis of tandem segments ofpoIC to produce and select segmentally mutated enzyme forms suitable for analysis of specific parameters governing dNTP analog binding and Pol function. The results of the latter experiments and experiments exploiting site-specific mutagenesis within the 1175-1273 Pol block should prove useful, even in the absence of a formal X-ray crystallographic analysis, in the development of a three-dimensional concept of the enzyme's Pol domaia.

ACKNOWLEDGEMENTS

The authors thank Dr. Nicholas Cozzarelli for providing B. subtilis polA59 his leu met; Dr. David Dubnau for providing the isogenic B. subtilis strains containing the respectivepolC22-27 mutations, and Dr. Julian Gross for providing B. subtilis strain 2355 used in the analysis ofmut-1. This work was supported by NIH grant GM28775 to N.C.B.

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49 Reha-Krantz, L., Stoeki, S., Nonay, R., Dimayuga, E., Goodrich, L., Konigsberg, W. and Spicer, E.: DNA polymerization in the absence ofexonucleolytic proofreading: in vivo and in vitro studies. Proc. Nat. Acad. Sci. USA 88 (1991) 2417-2421. Sanjanwala, B. and Ganesan, A.: DNA polymerase Ill gene o f Bacillus subtilis. Proc. Natl. Acad. Sci. USA 86 (1989) 4421-4424.

Sanjanwala, B. and Ganesan, A.: Genetic structure and domains of DNA polymeraseIll of Bacillus subtilis. Mol. Gen. Genet. 226 (1991) 467-472. Tomasiewicz, H. and McHenry, C: Sequence analysis of the Escherichia coil dnaE gene. J. Baeteriol. 169 (1987) 5735-5744.