THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 20, Issue of May 20, pp. 14655-14660, 1994 Printed in U.S.A.

Amino Acid Sequence Motifs Essential to 3’ + 5’ Exonuclease Activity of Escherichia coli DNA Polymerase 11*

(Received for publication, January 24, 1994)

Yoshizumi Ishino$, Hiroshi IwasakiO, Ikunoshin Kato, and Hideo ShinagawaO From Biotechnology Research Laboratories, Takara Shuzo, Otsu, Shiga 520-21, Japan and the §Department of Experimental Chemotherapy, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan

Many DNA polymerases have conserved sequences re- quired for 3’ 4 5’ exonuclease activity, which contrib- utes to the accuracy of DNA replication by removing misincorporated nucleotides prior to chain elongation. Using amino acid sequence alignments, we predicted the putative active site of the 3’ + 5‘ exonuclease of Escherichia coli DNA polymerase 11. Site-directed mu- tagenesis at Dl554 E1674 D155A/E157& D2284 Y330F, and D334A, which are in the predicted exonuclease ac- tive regions, specifically inactivated 3‘ + 5’ exonucleo- lytic activity but not DNA-polymerizing activity of E. coli DNA polymerase 11. Furthermore, all of the mutants were diminished in the in vitro proofreading ability, as judged by their increased insertion and extension of wrong nucleotides.

These findings indicate that the 3‘ 4 5‘ exonuclease region of the E. coli DNA polymerase I1 is in the amino- terminal part of the protein, as it is in other DNA polym- erases, and are consistent with the proposal of an evo- lutionary conserved 3‘ + 5’ exonuclease active site in most DNA-dependent DNA polymerases of both prokary- otic and eukaryotic origin by Bernad et al . (Bernad, A, Blanco, L., Lazaro, J. M., Martin, G., and Salas, M. (1989) Cell 59,219-228).

The bacterium Escherichia coli has three DNA polymerases (1). DNA polymerase I (pol I),’ encoded by polA, is involved in DNArepair (2) and replication (3). The DNApolymerase I11 (pol 111) holoenzyme constitutes the replicative polymerase in the organism (4). The a-subunit of the holoenzyme encoded by dnaE (poZC) has the DNA-polymerizing activity. However, no biological role has yet been assigned to DNA polymerase I1 (pol II), which is encoded by the polB gene. The polB mutants iso- lated to date are not defective in DNA replication or repair (5-7).

pol I1 was originally purified, and its 3‘ + 5’ exonuclease activity was detected by Wickner et al. (8). Recently, we and others have cloned the polB gene and analyzed its nucleotide sequence (9-13). To date, primary structures of many DNA polymerases have been revealed from the nucleotide sequences of the cloned genes, and extensive comparisons about the rela- tionship between the structure and the functions have been discussed. From the multiple alignment based on their se- quences, some classifications of DNA polymerases into two ma- jor groups, E. coli pol I family and the eukaryotic DNA polym- erase a family, have been proposed (14, 15). The deduced

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: -1.: 81-775-43-7215; Fax: 81-775-43-2494.

The abbreviation used is: pol, polymerase.

amino acid sequence, to our surprise, showed that pol I1 shares homologies with eukaryotic DNA polymerases including hu- man DNA polymerase a rather than E. coli pol I or pol 111. pol I1 is the first example of a eukaryotic-type DNA polymerase in bacteria, and this finding jeopardized the apparent criteria of the previous classification of DNApolymerases into prokaryotic or eukaryotic categories. Recently, Ito and Braithwaite (16) have classified DNA polymerases into four families. Family Ais named after its homology to the product of the polA gene of E. coli; family B is named after polB of E. coli; family C is named after the polC (dnaE) gene of E. coli; and family X includes eukaryotic DNA polymerase p and terminal nucleotidyl trans- ferases and does not share homology with any families de- scribed above. Family B DNA polymerases are quite extensive in number and variety. Family B includes 1) eukaryotic cellular DNA polymerases; 2) viral DNA polymerases, such as those from herpesviruses (17-19) and T4 (20); and 3) the protein- primed DNA polymerases identified so far, either from bacte- riophages, as M2 (21) and $29 (22), from animal viruses as adenovirus (23), or from eukaryotic linear DNA plasmids as that from Kluyueromyces lactis plasmid pGKLl (24). Recently, DNA polymerases from archaeon such as Pyrococcus furiosus (251, Sulfolobus solfataricus (261, and Thermococcus litoralis (27) were found to be included in family B. Most of the family B DNA polymerases, if not all, are sensitive to aphidicolin for the DNA-polymerizing activity (281, which was one of the early criteria for whether the DNA polymerase is eukaryotic a-type DNA polymerase or not. The deduced primary structure of pol I1 is homologous to that of a-like DNApolymerases (111, and its DNA-polymerizing activity is inhibited by aphidicolin (29, 30). Most of this family of DNA polymerases contain the highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp, which has been suggested to form a part of the dNTP binding site. Experiments using mutant DNA polymerases of herpesvi- ruses (17,31,32) and $29 DNApolymerase (33-36) have shown that amino acid substitutions in this and other conserved se- quences in the carboxyl-terminal portion resulted in defects in the DNA-polymerizing activity without affecting the associated 3’ + 5’ exonuclease activity.

Many DNA polymerases are known to have an associated 3’ + 5‘ exonuclease activity, which contributes to the accuracy of DNA replication by hydrolysis of misincorporated nucleoti- des prior to chain elongation (37). Based on amino acid se- quence alignments, involving family A and B DNA polym- erases, and site-directed mutagenesis studies, it was proposed that the 3‘ - 5‘ exonuclease active site of prokaryotic and eukaryotic DNA polymerases is located in an amino-terminal domain, as occurs in E. coli pol I, being particularly formed by three conserved amino acid segments, named Exo I, Exo 11, and Exo I11 (38). These segments contained the critical residues involved in metal binding and 3‘ + 5’ exonucleolytic catalysis of pol I, identified by crystal structure and mutational analysis (39-41). The Exo I segment, wrongly identified in the case of

14655

14656 3' + 5' Exonuclease of E. coli DNA Polymerase 11

bacteriophage T4 and eukaryotic viral and cellular DNA polym- erases was further corrected (42-44), and the new proposal was supported by mutational analysis in DNA polymerase I1 (43) and DNA polymerase I11 (42) of Saccharomyces cerevisiae. Other mutational analyses indicate that the three regions, Exo I, Exo 11, and Exo 111, are critical for the exonuclease activity (45-48).

The amino-terminal portion of pol I1 aligns well with these conserved Exo I, Exo 11, and Exo I11 regions (see Fig. l), and all conserved amino acids, Asp"', G ~ u ' ~ ~ , Asp228, W 3 0 , and Asp334, in the segments are present, which correspond to the critical residues for the 3' + 5' exonuclease activity of pol I. In this study, we made mutant pol 11s with the amino acid substitu- tions in these residues predicted to be important for the exo- nuclease activity (see Fig. 1) and showed that these residues were indeed essential for the 3' "-f 5' exonuclease activity.

MATERIALS AND METHODS Materials-Enzymes for in vitro manipulations of DNA and the site-

specific mutagenesis kit (Mutan-K) were the products of Takara Shuzo (Kyoto, Japan). [a-32PldCTP and [methyl-3HllTP were purchased from Amersham Japan (Tokyo, Japan). E. coli BL21(DE3) and plasmid pET8c (49) for overproduction of wild type and mutants of DNA polym- erase I1 were kindly provided by Dr. F. Studier. Oligonucleotides for mutagenesis and sequencing were synthesized using an autosynthe- sizer 380B (Applied Biosystems, Foster City, CA).

Plasmid Constructions-To produce DNA polymerase I1 more e a - ciently than previously (29), we constructed another expression plasmid using the PET system (49). The 5"region of polB was cut out from the runaway plasmid pTH116 (9) and inserted into M13 mp18. Using the resulting recombinant single-stranded phage, mppolB1, a site-specific mutagenesis was done to change the initiation codon from GTG to ATG and make an NcoI site (4CATGG) at the translation initiation site of the polB gene. The oligonucleotide primer was dGCCTGCGCCATGGT- GAAAATCC, and site-specific mutagenesis was carried out by the method of Kunkel et al. (50) using Mutan-K. The entire polB gene was reconstructed by returning the fragment containing the ATG codon in the unique NcoI sequence onto the plasmid pTH3055 (9) using EcoRI and AccI and then transferring a 4-kilobase NcoI-Hind111 (blunted) fragment containing the polB coding region onto the NcoI-BamHI (blunted) site of PET-8c plasmid, resulting in pTH128. For the construc- tion of DNA polymerase I1 mutants, the NcoI-HincII fragment contain- ing a part ofpolB was cut out from pTH128 and inserted into M13mp18 (mppolB2), and site-specific mutagenesis was done with Mutan-K. Oli-

TATAGAAAC (D155A), dGCGGGTGGlTGCAATATCTA (E157A), gonucleotide primers used for mutagenesis were dGTTTCAATAGC-

GCAGAGCGAACTGCAC (D228A), dTTCAGGTTAA4AGlTGCCAG (Y330F), and dAGCTCGCAAGC'MTCAGGTT (D334A). After site-spe- cific mutagenesis, the NcoI-HincII fragments were exchanged with an NcoI-HpaI fragment of the pTH128, resulting in pTH129, pTH130, pTH131, pTH132, pTH133, pTH134, and the nucleotide sequences of the entire polB genes were confirmed by using sets of specific primers synthesized by an autosynthesizer (Applied Biosystems, 380B). These resulting plasmids were introduced into E. coli BL21(DE3), and DNA polymerases were induced by adding isopropyl-1-thio-P-D-galactopy- ranoside.

Purification of Wild Qpe and Mutant Enzymes of DNA Polymerase II-E. coli BL21(DE3) carrying pTH128, -129, -130, -131, -132, -133, or -134 was grown at 37 "C with shaking in 1 liter of L broth (10 giliter bactotryptone, 5 g/liter yeast extract, 5 g/liter NaC1) containing 100 pg/ml ampicillin. When the culture reached A, = 0.5, isopropyl-l-thio- p-D-galactopyranoside was added to a concentration of 1 m~ for the induction of pol 11, and incubation was continued for another 3 h. The crude extract was obtained by the procedure described earlier (29). The cells were harvested, washed with a buffer containing 50 mM Tris-HC1, pH 7.5, and 10% sucrose, and resuspended in 20 ml of the same buffer with 15 m~ spermidine. Egg white lysozyme was added to a final con- centration of 0.2 mg/ml and incubated at 4 "C for 1 h. Triton X-100 was added to a concentration of 1% (v/v) and the mixture was gently shaken at 37 "C for 5 min. After centrifugation of the mixture a t 23,000 x g for 1 h, 20 ml of the crude extract was obtained. After ammonium sulfate precipitation, the pol 11-containing fraction was dialyzed against buffer A containing 50 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 7 mM 2-mercapto- ethanol, and 10% glycerol. The dialysate was applied to a MonoQ 10/10

dGCGGGTGGTTGCAATAGCTATAGAAAC (D155AiE157A), dCATTC-

column pre-equilibrated with buffer A, using a fast protein liquid chro- matography system (Pharmacia LKB Biotechnology Inc.). After the col- umn was washed with buffer A, it was developed with a 200-ml linear gradient of 0-0.5 M NaCl in buffer A. The pol 11-containing fractions were eluted at 0.22 M NaCl as a sharp peak. The pooled fraction was dialyzed against buffer B containing 10 m~ potassium phosphate (pH 6.81, 7 m~ 2-mercaptoethanol, 0.05 mM CaCl,, and 10% glycerol and then applied onto a hydroxylapatite column (Koken, Tokyo, Japan) pre- equilibrated with buffer B, which was operated with a fast protein liquid chromatography system. After washing with buffer B, the column was developed with a 30-ml linear gradient of 10-200 m~ potassium phosphate. The pol I1 fraction was eluted at 140 mM potassium phos- phate as a single sharp peak. After the pol I1 fraction was dialyzed against buffer A, the dialysate was applied to a Mono S HR 5/5 column (Pharmacia). The column was washed with buffer A and developed with a 12-ml linear gradient of 0-0.3 M NaC1. The pol I1 activity was eluted at 0.2 M NaCl as a very sharp single peak.

Assay of DNA-polymerizing Activity-DNA-polymerizing activity was assayed as described by Ishino et al. (29). One unit is defined as the amount of enzyme catalyzing the incorporation of 10 nmol of dTMP per 30 min at 37 "C into activated calf thymus DNA.

Assay of Exonucleolytic Actiuity-The 3' - 5' exonuclease activity was assayed essentially as described by Ishino et al. (29). Instead of the 302 base pairs of PuuII fragment of pUC19, the 220-base pair AuaII fragment of pUC19 was labeled at the 3'-ends by a filling reaction with Bacillus caldotenaz DNA polymerase in the presence of [CY-~~PI~CTP. The reaction mixture (50 p1) containing 50 m~ Tris-HC1 (pH 7.5), 10 mM MgCl,, 5 m~ 2-mercaptoethanol, and 80,000 cpm of labeled DNA was incubated at 37 "C, and then radioactivity in the ethanol-soluble frac- tion was measured by Cerenkov radiation. One unit is defined as the amount of enzyme catalyzing the excision of 210 fmol of dCMP under the conditions described above. From this calculation, 1 unit of exo- nuclease activity of wild type pol I1 corresponds to 1 unit of polymerase activity for the wild type protein.

In Vitro Misincorporation Assay-A 17-deoxynucleotide primer and a 37-deoxynucleotide template were synthesized, the sequences of which were the same as those used by Reha-Krantz et al. (46). The primer oligonucleotides were labeled with [y-32PlATP and polynucleotide ki- nase prior to annealing to the template. Primer extension reactions were carried out in 20 pl of the reaction mixture containing 10 mM Tris-HC1 (pH 7.5), 6 mM MgCl,, 20 m~ KCl, 0.5 p~ template-primer DNA, and 1 unit of pol 11. For complete reaction, 100 p~ each of dATP, dGTP, dCTP, and dTTP were added and dGTP was omitted for misin- corporation assay. After incubation at 37 "C, reactions were stopped by adding stop solution containing 95% formamide, 0.05% bromphenol blue, and 0.05% xylene cyanol, and 1.5 p1 of each mixture was applied onto a 20% polyacrylamide gel containing 8 M urea. Electrophoresis was done with a constant wattage of 40.

Comparison of Primer Extension Abilities-Primer extension reac- tions were carried out in 15 1.11 of the reaction mixture containing 10 mM Tris-HC1 (pH 7.5), 6 m~ MgCI,, 1 pg of MI3 mp18 single-stranded DNA, 2 pmol of rhodamine-labeled primer (R-dG'ITITCCCAGTCACGAC), 1 mM dNTPs, and 2 units of wild type pol I1 or the mutant enzymes. Two microliters of each mixture was applied onto a 6% polyacrylamide gel containing 8 M urea. The sequencing ladder of MI3 mp18 was loaded as a size marker. Electrophoresis patterns were imaged by scanning gel plates by a fluorescent image analyzer FMBIO (Takara Shuzo) as de- scribed earlier (51).

RESULTS AND DISCUSSION

Prediction of Amino Acids Required for 3' + 5' Exonuclease Activity of DNA Polymerase 11-We added the amino acid se- quence of pol I1 to the computer alignment performed by Mor- rison et al. (43) and found that pol I1 has three conserved regions in the amino-terminal part of the protein (Fig. 1). Three conserved regions for the 3' + 5' exonuclease were originally proposed by Salas' group (38). The FDIET segment corresponds to their corrected Exo I region (44). In the FDIET region, Asp3" and G ~ u ~ ~ ~ of pol I (which correspond to Asp"' and to G ~ u " ~ of pol 11, respectively) are the only invariant residues. Asp424 (cor- responding to Asp228 of pol 11) in Exo 11, and T y r A S 7 and Asp601 (corresponding to W 3 0 and Asp334 of pol 11) are also very well conserved. Amino acid substitutions of these residues result in a defect in the 3' + 5' exonuclease activity of pol I (40). Other mutations resulting in deficiency of the 3' + 5' exonuclease are

3' + 5' Exonuclease of E. coli DNA Polymerase II 14657

Exol (FDIt3)

HSV E.C POI I1

VZV E I V CMV S.C. POI 111

ACMNPV S.C. POI I1

AdWWViNS Vacflnia T4 029 eBLIl_ S.S. POI 7 E.C. Pal Ilk B I Pol 111 SPO2 T5 Tl E.C POI I

- Ex011

E.C. POI I1

H.s. Pol D

s.c POI I

D.m. Pol (i

vzv EBV CMV s.c Pal 111 s.c POI I1 AcMNPV T4 AdmWINr vacf1n1a 029 PRDl S.C. P o l l

8.S POI 111 E.c Pol 1111

T5 SPO2

l7 E.C POI I

nsv

219.235

634-650 635-651

462-478 660616

443459 375.391 4M-420 396.412 374490 276-292 210.226 271.287 234-250 58.73

222-237 67.83

502.517 95-110

180-205 70.85

416-431 56-72

V 0 Y M

D E N H

G -

n

- Exolll

nsv E,c. Pol I1

VZV

CMV E BV

S e POI 111

AcMNPV S.C. POI I1

AdenDYlNS Vaccinia 14

PRDl 029

S.C. POI 1

SPO2 E.c POI Ilk

T5 T l E.c. Pol I

T A S K EO

sequence alignments are based on Morrison et al. (43). The residues corresponding to those of the 3' + 5' exonuclease active site of E. coli DNA FIG. 1. Alignments of DNA polymerase amino acid sequences containing conserved 3' + 5' exonuclease active residues. The

polymerase I are indicated in white lettering. Prominently conserved sequences are boxed. Above the line are aphidicolin-sensitive DNA polym- erases. HSV, herpes simplex virus; VZV, varicella zoster virus; EBV, Epstein-Barr virus; CMV, human cytomegalovirus; AcMNPY Autographa California nuclear polyhedosis virus; S.C., Saccharomyces cereuisiae; B.s., Bacillus subtilis; H.s., Homo sapiens; D.m., Drosophila melanogaster; E.c., E. coli.

the following: residues Asp' and Glu7 of T7 DNA polymerase in Exo I motif (45); Asp" and Glu14 in Exo I motif; Asp66 in Exo I1 (38); T y P 5 and Asp169 in Exo I11 (47) of $29 polymerase; Aspzgo and GluZg2 in Exo I motif of yeast DNA polymerase I1 (43); Asp324 in Exo I11 (46) and Aspz4' in Exo I1 (48) of T4 DNA polymerase; Asp321 and Glu323 of Exo I motif; and Asp355 in Exo I1 of yeast DNA polymerase I11 (42). These amino acid residues correspond to critical residues of pol I described above. There- fore, we predicted such amino acid residues as Asp155, Glu15', Aspzz8, and Asp334 of pol I1 to be important for the 3' "-f 5' exonuclease activity. Then, by in vitro site-specific mutagen- esis, we constructed the six mutant polB genes that produced altered pol I1 enzymes with amino acid substitution of D155A, E157A, D155A/E157A, D228A, Y330F, and D334A. The D155A/ E157A is a double mutant within the Exo I motif.

Overexpression and Purification of pol II-In the previous work we constructed an overproducing system of pol I1 by in- troducing the polB gene with its own promoter into a runaway plasmid (29). To produce the polymerase more efficiently, we constructed another expression plasmid using the PET system developed by Studier (49). By the T7 expression system, we were able to establish a simple and rapid procedure that leads easily to homogeneous enzyme (more than 99% punty by pho- tographic densitometer) in good yield. Mutant pol I1 proteins could be purified by the same procedure as wild type protein. The retention times of the mutant proteins in each fast protein liquid chromatography column were the same as those of wild type protein. The details of the purifications will be published elsewhere.' Five hundred micrograms of the wild type and mu- tant pol I1 proteins on an average were obtained from 1 liter of culture.

3' + 5' Exonuclease Activity of the Mutant pol Ils-All highly purified mutant pol 11s were assayed in their DNA-polymeriz- ing and 3' + 5' exonucleolytic activities. Table I summarizes the effects of each mutation on the polymerizing and exonu- cleolytic activities of the pol 11. All the mutant enzymes had almost the same specific activities of DNA polymerization as the wild type pol 11, whereas the mutants showed a large de- crease in the 3' + 5' exonuclease activity and the exonuclease/ polymerase ratios were 2-4 orders of magnitude lower than that of the wild type enzyme. The E. coli BL21 (DE3) that we used for overexpression of the mutant polB genes has a wild type polB in its chromosomal DNA. However, we purified all of

Y. Ishino, H. Iwasaki, T. Honuchi, I. Kato, and H. Shinagawa, manu- script in preparation.

TABLE I Effects of E. coli DNA polymerase II mutations on 3' - 5'

exonuclease activity The 3' - 5' exonuclease activity was measured on double-stranded

DNA.

Mutation DNA 3' - 5' polymerase exonuclease (Exolpol)

Ratio

unitslmg units f mg

Wild type 5.8 X 105 5.8 x IO6 1 D155A 3.7 x 105 5.0 x 10' E157A D155AiE157A D228A Y330F D334A

1.4 x 10-4 2.6 x 105 2.1 x 102 8.1 x 10-4 4.4 X 105 3.7 X 105 3.5 X 105 4.6 x 105

5.8 x 103 1.7 x 9.7 x 103 2.1 x 10-2

2.1 x 102 4.8 x 10-4 2.9 x 10' 7.8 x lo4

the pol I1 protein with exactly the same procedure in parallel. Therefore, we concluded that the contamination of the exo ac- tivity from the wild typepolB was less than of the wild type enzyme and that the 100-fold reduced activity is due to the residual activity of the mutant pol 11.

Deficiency of in Vitro Proofreading Activity of Mutant pol Ils-To test if the 3' "-f 5' exonuclease activity works for proof- reading, an in vitro misincorporation assay was done. As shown in Fig. 2, wild type pol I1 could add only two nucleotides in the absence of dGTP. However, mutant proteins could efficiently incorporate incorrect substrates other than dGTP at the 3rd, 5th, and 6th positions. From this result, it can be predicted that the 3' + 5' exonuclease activity is responsible for correction of nucleotides misincorporated by pol 11. It is not known why pol I1 has an associated 3' "-f 5' exonuclease activity and how the activity works in the cell. We showed that the expression of the polB is regulated by the SOS system (9). This fact suggests the possibility of the involvement of pol I1 in DNA repair, recombi- nation, and mutagenesis. I t would be necessary to define the concrete role of pol I1 in vivo to understand the meaning of the existence of the 3' + 5' exonuclease activity in pol 11.

The reason why the elongation ability of the mutant D334A, the exonuclease activity of which is higher than other mutants, looks strongest of all six mutant enzymes (Fig. 2, lanes 21-23) is not known.

Arrest of DNA Synthesis by pol II-By using "in vitro" primer extension experiments, it has been shown that DNA polym- erase I1 is arrested at specific sites on natural DNA templates (52). Although the barriers are thought to be sites of secondary structure, the arrest of strand synthesis cannot be explained by the palindromic sequence alone, and the natural arrest sites may have some biological significance (53). The analysis of the

14658 3' + 5' Exonuclease of E. coli DNA Polymerase 11

E VI VI

FIG. 2. In vitro misincorporation as- say. Reaction products of the primer ex- tension using the primer-template shown at the right, with wild type and six mu- tants of pol 11, were analyzed on 2 0 6 poly-

Lanes 1 and 2.5, "P-labeled primer (17 acrylamide gels containing 8 M urea.

bases); lanes 2 and 24, :'*P-labeled tem- plate (37 bases); lanes 3, 6. 9, 12, 15, 18, and 21, products from complete reactions using all four dNTPs for 20 min; lanes 4, 7, 10, 13, 16, 19, and 22, products from reactions without dGTP (misincorpora- tion assay) for 5 min; lanes 5.8, 11, 14, 17, 20, and 23, misincorporation assay for 20- min reaction.

I 2 3 4 S 6 7 8 V IO I I 12 13 I4 IS 16 17 18 19 20 21 22 21 I4 2 5 nn nnnnn

37 + - 0

DNA elongation ability of pol I1 in vitro may help to understand some function of pol I1 in vivo. We used M13 single-stranded DNA as template and a 5"terminal labeled oligonucleotide as primer. We found that pol I1 has one major arrest site around 150 bases from the 5' terminus of the primer (#6326 in Fig. lA of Ref. 541, where the sequence does not show the possibility of the palindromic structure with high stability (Fig. 3a) . It is not known why the area works as a bamer. By contrast, all of the six exonuclease mutant enzymes could pass through the ar- rested position and extend the strand further (Fig. 3b) . In the same experiment, T4 DNA polymerase was found to be arrested at the same site as that of pol I1 (Fig. 3b, lane 8). This site may be common for a-like DNA polymerases. I? furiosus DNA polym- erase, which is included in Family B, could pass through it (Fig. 3b, lane 12). This may be due to the resolution of the secondary structure at the high reaction temperature (75 "C) used in the case ofI? furiosus DNApolymerase. pol I-like DNApolymerases such as Klenow fragment, B. caldotenux DNA polymerase, and Thermus aquaticus DNA polymerase were not arrested at the site (Fig. 36, lanes 9-11 ). Mutant pol I1 may incorporate some

5' I

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

+5 c + 4 T +3 c +2 A + I A T

0 A * T T-A A - T T-A C-G A - T C O G TmA C*G

Go C A. T

C*G A - T To A A * T Am T T*A I I 5' 3'

nucleotide at the arrested site that can help to continue strand elongation, and therefore, it would be interesting to analyze the sequence of the synthesized strand. The proofreading activity appears to be correlated with the processivity of the DNA po- lymerases. The bamer si te may form such a structure as to suppress the correct base pairing, and a s a result, i t causes incorporation and subsequent excision of nucleotide idling by the wild type pol I1 (55).

Active Site of 3' "+ 5' Exonucleolytic Activity of DNA Polym- erases-In the present study, using the amino acid sequence alignments, we predicted the 3' + 5' exonuclease active site of pol I1 and constructed six kinds of mutant polB genes and purified these mutant pol I1 proteins. The DNA polymerizing and exonuclease activities of all these mutant proteins were measured. All the mutants reduced only exonucleolytic activity, which indicates that exonuclease and polymerase domains are separated in pol I1 protein at least at the primary structure level. Thus, we concluded that five pol I1 residues, which are in the predicted Exo I, Exo 11, and Exo 111, have an important role for the 3' - 5' exonuclease activity but not for DNA-polymer-

3' + 5' Exonuclease of E. coli DNA Polymerase 11 14659

FIG. 3. Comparison of the chain elongation ability of DNA polym- erases on primed ssMl3 template. a. DNA polymerase reactions were done with 2 units of wild type pol 11. An equal amount of the reaction mixture was sampled a t 2 min (lane 1 ), 5 min (lane 2). 10 min (lane 3 ) . 20 min (lane 4 ) , and 30 min (lane 5). and stop solution was added. h, DNA polymerase reactions were done with 2 units of one of the polymerases under standard conditions. h n e s 1-7, wild type and mutants of pol I1 for 20 min ( I , D155A; 2, E157A. 3, D155AIE157A; 4, D228A; .5, Y330F; 6, D334A; 7. wild type); lane 8, T4 pol (Takara Shuzo) for 20 min; lane 9, Klenow fragment (Takara Shuzo) for 20 min; lane IO. R. cnldotrnnr pol (56) for 1 min; lane 11, 7: aquaticus pol (Per- kin-Elmer) for 5 min; lane 12, F? firriosus pol (25) for 5 min.

f - 2 0 0

4- 200

f 1 5 0

izing activity. Three of these five DNA polymerase I1 residues, Asp'" and GIU''~ (Exo I), and Asp"' (Ex0 II), were the most critical, in agreement with the results obtained by mutational analysis in the corresponding residues of pol I (40) and 629 DNA polymerase (38). On the other hand, DNA polymerase I1 residue QP" (Exo 111) has only a secondary role in exonucleo- lytic catalysis, again in agreement with mutational analysis of the corresponding tyrosine residue of pol I (40) and 629 DNA polymerase (47). Interestingly, whereas change of the con- served aspartate in the Exo I11 segment in Klenow (D501A) or 429 (D169A) gave rise to 3' 5' exonuclease inactivation (a reduction by 4 orders of magnitude), a similar mutant in DNA polymerase I1 (D334A) retained 2% of its intrinsic 3' -., 5' exonucleolytic activity.

Taking into account that each of these acidic residues is proposed to be important for metal binding at the 3' -> 5' exonuclease active site (40), the differences observed in the

those of pol I and (h29 DNA polymerase may suggest some different mode of metal binding. However, although the ternary structure around the active site of each DNA polymerase may be slightly different, our results support the proposal (33, 38) that the 3' -+ 5' exonuclease active site is evolutionarily con- served in prokaryotic and eukaryotic proofreading DNA pol-ym- erases.

Acknowledgments-We thank Dr. Akio Sugino for valuable discus- sion, and Drs. Margarita Salas and Luis Rlanco for critically reading the manuscript. We thank Kazuko Nakanishi for preparing the figures.

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2. Gross. J . . and Gross. M. 119159, ,%turn 224, Ilfifi-llfiR 3. Joyce. C. M.. and Grindley. N. D. F. (19H.l I .J. k I r f P m J l . 168. 636443 4. McHenry, C. S. I19RR)Annu. Ret'. 8rorhr.m. 57, 519-550 5. Camphell. J . L.. S o l l . L.. and Richardson. C . ('. I 14721 I'm. Nof l . Arori Srr

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