Proc. Nati. Acad. Sci. USAVol. 88, pp. 9473-9477, November 1991Biochemistry

Eukaryotic DNA polymerase amino acid sequence required for 3' ->5' exonuclease activity

(yeast DNA polymerase li/mutator phenotpe/proofreading/protein domain/DNA replication)

ALAN MORRISON*, JULIETrE B. BELL, THOMAS A. KUNKEL, AND AKIO SUGINOLaboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709

Communicated by Thomas A. Steitz, July 22, 1991 (receivedfor review May 20, 1991)

ABSTRACT We have identified an amino-proximal se-quence motif, Phe-Asp-lle-Glu-Thr, in Saccharomyces cerevi-siae DNA polymerase II that is almost identical to a sequencecomprising part of the 3' -- 5' exonuclease active site ofEscherichia coli DNA polymerase I. Similar motifs were iden-tified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3' -*5' proofreadingexonuclease activity. Substitution of Ala for the Asp and Gluresidues in the motif reduced the exonuclease activity ofpartially purified DNA polymerase II at least 100-fold whilepreserving the polymerase activity. Yeast strains expressing theexonuclease-deficient DNA polymerase Il had on average abouta 22-fold increase in spontaneous mutation rate, consistent witha presumed proofreading role in vivo. In multiple amino acidsequence alignments of this and two other conserved motifsdescribed previously, five residues of the 3' -* 5' exonucleaseactive site ofE. coiUDNA polymerase I appeared to be invariantin aphidicolin-sensitive DNA polymerases known to possess 3'- 5' proofreading exonuclease activity. None ofthese residues,however, appeared to be identifiable in the catalytic subunits ofhuman, yeast, or Drosophila a DNA polymerases.

Class B DNA polymerasest are related through a series ofconserved amino acid sequences that occur in the order IV,II, VI, III, I, V, with region I being the most highly conserved(2). Regions II, III, I, and V are implicated in DNA poly-merase functions (3-8). The location of the 3' -* 5' exonu-clease activity domain has been sought through amino acidsequence similarity with the apparently unrelated E. coliDNA polymerase I. Crystallographic and mutational evi-dence has determined that the 3' -*5' exonuclease active siteof E. coli DNA polymerase I is composed of a group ofcarboxylate residues (D355, E357, D424, and D501) cluster-ing around two metal ions that coordinate the 3'-terminalphosphate and a second group of residues (L361, F473, andY497) located around the terminal base and ribose positions(9, 10). It has been proposed that residues D355 and E357 areconserved in the left part of region IV ofaphidicolin-sensitiveDNA polymerases (11-13). Similarly, residues D424, Y497,and D501 were suggested to be conserved in the right part of,and distal to, region IV (4, 12, 13). Mutation to alanine ofthree of the relevant carboxylate residues in bacteriophage029 DNA polymerase leads to exonuclease-deficient DNApolymerase activity (13). Though related to other aphidicolin-sensitive DNA polymerases, 429 DNA polymerase does nothave a well-conserved region IV (2) and may not be a goodtest case for alignments involving this region. With coliphageT4 DNA polymerase, which does have a well-conservedregion IV (2, 11, 12), mutation to alanine ofresidues D189 andE191, predicted to correspond to the E. coli polymeraseresidues D355 and E357, does not lead to exonuclease

deficiency, calling into question the concept of a generallyconserved 3'-* 5' exonuclease active site (14).We present evidence that another amino acid sequence

motif, typically occurring about 65 residues amino-proximalto region IV, corresponds to the E. coli DNA polymerase Isequence containing residues D355 and E357. Mutation ofthecorresponding carboxylate residues in Saccharomyces cere-visiae DNA polymerase Ilt resulted in the predicted exonu-clease-deficient polymerase activity in vitro and spontaneousmutator phenotype in vivo. In addition, our amino acidsequence alignments support the earlier proposals (4, 12, 13)that E. coli DNA polymerase I residues D424, Y497, andD501 are also conserved in aphidicolin-sensitive DNA poly-merases, with the important exception of a DNA polymer-ases.

MATERIALS AND METHODSYeast Strains. CG379 (MATa his7-2 adeS-J leu2-3,-J12

trpl-289 ura3-52 CANJ), AMY52-3D (MATa ura3-52 leu2-1lysJ-J ade2-1 hisl-7 hom3-10 trpl-289 canl), and BJ3501(MATa pep4::HIS3 prbAL1.6 his3-A200 ura3-52 can] gal7)were used.Plasmid Construction. The genetic code for the amino acid

sequence FDIET was altered to that for FAIAT by site-directed mutagenesis (16) using the synthetic oligonucleotide5'-ATGGCATTTGCTATAGCAACCACGAAG-3' (corre-sponding to nucleotides 1450-1476) and a 2.1-kilobase (kb)Pvu I-EcoRI fragment of POL2 DNA (17) cloned intoM13mpl8. The DNA was sequenced to verify the desiredalteration and cloned as a 2.1-kb Hpa I-EcoRI DNA fragmentinto the Nru I-EcoRI interval of plasmid YIp5, creating the6.7-kb plasmid YIpJB1.

Construction ofpoi24 Strains. The allele ofPOL2 in whichthe genetic code for the amino acid sequence FDIET isaltered to that for FAIAT is named pot24. Yeast cells weretransformed to Ura' with YIpJB1 DNA linearized usingBamHI to target integration to the POL2 locus (18). Southernblotting of DNA from transformants showed the expected12-kb Hpa I DNA fragment when probed with 32P-labeledPOL2 0.9-kb HindIII fragment, whereas wild-type yeast gavethe expected 5.3-kb Hpa I fragment. Integrants of BJ3501

*To whom reprint requests should be addressed.tDNA polymerases with sequence similarity to Escherichia coliDNA polymerase I are class A, and those with sequence similarityto eukaryotic DNA polymerase a are class B (1). We also refer toclass B DNA polymerases as "aphidicolin-sensitive" DNA poly-merases. The term "a-type" is not used, since it might causeconfusion with the different classification of DNA polymerases asa, f, y, 8, or E.

tS. cerevisiae DNA polymerases II and III are comparable tomammalian DNA polymerases e and 8, respectively (15). We retainthe names S. cerevisiae DNA polymerases II and III, since homol-ogy-rather than mere analogy-with mammalian DNA polymer-ases E and 8 has not been demonstrated. S. cerevisiae DNApolymerase I is, however, also referred to as S. cerevisiae DNApolymerase a.

9473

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

9474 Biochemistry: Morrison et al.

were maintained in medium lacking uracil. For phenotypicmeasurements, integrants of CG379 and AMY52-3D weregrown on 5'-fluoroorotic acid to select for excision of URA3(19) and reconstitution ofthe original genetic configuration asdetermined by Southern blotting and DNADNA hybridiza-tion. Yeast DNA from the resulting derivatives was amplifiedby the polymerase chain reaction using primers 5'-ATTCCAATCAGTTATTCGAGGCCAG-3' (nucleotides1175-1199) and 5'-CACCATTGAAGGTGGATATAA-CAGT-3' (complement of nucleotides 1729-1705) and se-quenced using primer5'-GTAGAAGCGCCACTTCATCG-3'(complement ofnucleotides 1669-1650) to determine whetherthe nucleotide sequence was that expected for the P0L2 orpoI24 alleles.

Spontaneous Mutation Rates. Spontaneous mutation rateswere measured as reversion of auxotrophs to prototrophyusing the Leningrad test (20) or as forward mutation tocanavanine resistance using the method ofthe median (20). Inthe former case, yeast cells grown in YPDA medium (26) tostationary phase were washed, suspended in water at densi-ties of lw106 to 107 cells per ml, and applied to plates using amultipronged replicator. For each determination, 500-600compartments were replicated onto plates containing syn-thetic medium either lacking or containing a limiting amountof the required amino acid. Plates were incubated for 5-10days at 30°C, and spontaneous reversion rates were calcu-lated by the P0 method.Enzyme Purification. Strain BJ3501 and its YIpJB1 inte-

grant expressing the pol24 allele were grown in YPDAmedium. Cells were disrupted and subjected to S-Sepharose

Proc. Natl. Acad. Sci. USA 88 (1991)

and Mono Q column chromatography as described (17, 21),except that the S-Sepharose column was eluted with buffercontaining 0.5 M KCl. The DNA polymerase II peak fromMono Q was purified by Mono S column chromatography.DNA polymerase II was distinguished from other polymeraseactivities by its characteristic elution positions from Mono Qand Mono S columns (21) and by assay under conditionsspecific for DNA polymerase II using high salt, low pH, andpoly(dA)*oligo(dT) as template-primer (17). The 3' -- 5'exonuclease activity of DNA polymerase II is described inHamatake et al. (21). Following the S-Sepharose step, DNApolymerase II is largely free of nonintrinsic nuclease activityand the ratio of polymerase to exonuclease remains approx-imately constant (21). This enabled us to measure exonu-clease conveniently in a single-strandedDNA nuclease assayby the release of acid-soluble radioactivity as described (21),except that reactions contained 0.1 M KCI and the DNAsubstrate was prepared as follows. Nicked, gapped calfthymus DNA (100 jig) was radiolabeled in a reaction con-taining 50 ,uCi of [a-32P]dTTP (1 Ci = 37 GBq), 10 units of E.coli DNA polymerase I Klenow fragment, 50 mM Tris-HCl(pH 7.5), 7 mM MgCl2, 1 mM dithiothreitol, 50 ,uM of eachdeoxynucleotide triphosphate, and 0.1 mg of bovine serumalbumin in a volume of 1 ml. Following incubation at 37C for2 hr, the DNA was purified twice by gel filtration on Seph-adex G-50 and heat-denatured before use.Amino Acid Sequence Alinment. We used the University

ofWisconsin Genetics Computer Group BESTFIT program ona Vax computer on sequences obtained from the NationalBiomedical Research Foundation and Swissprotein data

AHSVVzVEBVCMVS.c. Pol IIIS. c. Pol IIAcMNPVAdenovirusVacciniaT4*29

BH

364-399345-3802 92-323297-32 9317-34 728 6-3 16192-222137-14 6137-14 6108-117

8-17

E.c. Pol III E 8-17B.s. Pol III 421-430T5 134-143T7 1-10E.c. Pol I 351-360

* *

L C F D I E C K A G GLX L C F D I E C K S G GAL A F D I E C L G..CL S F D I E C M S..IM S F D I E C A G..

V M A F D I E T T K..LL C Y D I E T H S..F VT Y D V E T Y TYLF L D I E GH FVA N Cq D I EX T GM Y SCF D F E TT

E D E L A F P V AS N E L A F P D A. . E E G F P T A. G E G G F P C A. R I G V F P E P. P P L K F P D S. D G H NM S K A

G H P E D L V I Q I S C L L YT H L EID L V I Q I S C L L YT N E A D L I L Q I S C V L WE K S DIDI IV I Q IS C VFcYE . . Y D P V I Q IAN V V YA . . VID Q I M MI S Y M IDS. VDD IMS FVV

QV LD TET GY VIV F D V E T T GP VA F D S ETS AMI IIV S D IEE AN AVAIF D T|E TID S

CS. c. Pol I 616-634 V R L F N N E K A M L S CC C A K V E DE _D[ VH. s. Pol a 609-633 V E V A A T E R T L L G F F L A w H K I P D ID.m. Pol a 635-659 V H K H D S E R A LL S W F L A Q Y Q K I D A D LEj6V 437-461 V L E F D S E F E 4 L L A F M T L V K Q Y G E FVZV 418-442 V L E F D S E F E L I A F M Tt V K Q Y AWPEEBV 350-374 V Y E F P.S E L D M L Y A F F Q L I R D L S V ECMV 379-403 V Y E F P ,S E Y E L L L G F M L F Q R Y A P A FS. c. Pol III 371-395 I F S H A T E E E M L S N WR N [ I I K V D P VS.c. Pol II 349-373 I F N E N D E V A L L Q R F FE H I R D V R P VACMNPV 251-275 V M F Q N E I D M I T A F FDM I K I T N P DVI'~4 185-209 Y M P F D N E R D M L M E Y I N E Q K R P

FIG. 1. Alignments ofDNA polyrfierase amino acid sequences proposed to contain conserved 3' 5' exonuclease active site residues. Thesingle-letter code is used. Numbers refer to amino acid residues. Boxed are prominent identities or similarities scoring at least 0.5 on the Dayhoffmatrix as used in ref. 24. (A) Conserved region, beginning 54-109 residues amino-proximal to region IV (as defined in ref. 2), ofaphidicolin-sensitive DNA polymerases known to possess an associated 3' -* 5' exonuclease activity. Asterisks mark invariant Asp or Gluresidues proposed in this paper to be 3'-. 5' exonuclease active site residues. Sequences are aligned with E. coli DNA polymerase I and relatedenzymes in B as we propose in the text. (B) Sequence comprising part of the exonuclease active site, including residues D355 and E357, of E.coli DNA polymerase I aligned with sequences from related enzymes. (C) Left part of region IV of aphidicolin-sensitive DNA polymerases,previously proposed (11-13) to be similar to the 3' -*5' exonuclease active site sequences in B. Sequences in C and B are aligned as suggestedin refs. 11-13. [In ref. 13, the left part of region IV was proposed for most polymerases, but not for S. cerevisiae DNA polymerase I (see text),whereas for A. californica nuclear polyhedrosis virus and adenovirus DNA polymerases, the sequences given in A were proposed.] Pol, DNApolymerase; HSV, herpes simplex virus; VZV, varicella zoster virus; EBV, Epstein-Barr virus; CMV, human cytomegalovirus; AcMNPV, A.californica nuclear polyhedrosis virus; S.c., S. cerevisiae; E.c., E. coli; B.s., Bacillus subtilis; H.s., Homo sapiens; D.m., Drosophilamelanogaster; E. coli Pol III E, e subunit of E. coli DNA polymerase III.

Proc. Natl. Acad. Sci. USA 88 (1991) 9475

FDIET IV 11 1iii

BamHI

FDIET

A AIV

BamHI

Yl pJB1

FAIAT IV 11 III I V

URA3 pol2-4

FIG. 2. Structures ofPOL2, plasmid YIpJB1, and plasmid integrant. (Top) S. cerevisiae POL2 gene (open arrow) and the locations ofDNAencoding the amino acid sequence FDIET, conserved regions (defined in ref. 2) of aphidicolin-sensitive DNA polymerases (bold Romannumerals), and the BamHI site. Thick lines, flanking yeast DNA. (Middle) Plasmid YIpJB1 used to construct pol24, containing part of POL2with the code for the amino acid sequence FDIET changed to that for FAIAT (open box). Fine line, vectorDNA containing URA3 gene. (Bottom)Integration of YIpJB1 at the POL2 locus, resulting in duplication of the 5' region of POL2 and formation of pol24, which contains the codefor the mutated amino acid sequence FAIAT. Excision of the plasmid by homologous recombination (not shown) restores the original genomicDNA configuration leaving either POL2 or pol24, depending on whether the crossover occurs downstream or upstream, respectively, ofDNAencoding the FDIET or FAIAT amino acid sequences.

bases. Aphidicolin-sensitive DNA polymerase sequenceswere aligned with herpes simplex virus (type I, strain An-gelotti) DNA polymerase sequence to produce the alignmentsshown, with the following exceptions: yeast DNA polymer-ase II was aligned with yeast DNA polymerase III, Epstein-Barr virus, and Autographa californica nuclear polyhedrosisvirus DNA polymerases; adenovirus DNA polymerase wasaligned with herpes simplex virus DNA polymerase residues300-1235, except that adenovirus residues 435-445 werealigned visually; T4 residues 108-117 were aligned visually,

9

61M . 0.4 ---

E - 0.3 -la 2

D;0.2 -'

01 Z0.10

,Q4

6 10.3

10.2O 6

111 0.4 Z

15 20

Fraction number

FIG. 3. Mono S column chromatography of wild-type and mutantyeast DNA polymerase II. DNA polymerase II was partially purifiedfrom 12 liters of wild-type BJ3501 cells grown to OD600 = 2.6 or 24liters ofpol24 cells grown to OD600 = 1.4. Data points are the meansof three determinations.

and otherT4 sequence alignments are from refs. 2, 11, and 14.PRD1 DNA polymerase was aligned with 429 DNA poly-merase; alignment of )29 DNA polymerase is based on ref.13. Class A DNA polymerase amino acid sequences werealigned with E. coli DNA polymerase I sequence and/oralignments were taken from refs. 12, 13, 22, and 23.

RESULTS AND DISCUSSIONConserved Amino Acid Sequence Amino-Proximal to Region

IV. Computer-generated alignments of the amino acid se-quence of yeast DNA polymerase II with the sequences ofyeast DNA polymerase III and herpes viral family DNApolymerases identified a conserved region beginning 54-82residues amino-proximal to region IV (Fig. 1A). This region,previously identified in the latter set of DNA polymerases(25, 26), was not found in human, yeast, or Drosophila aDNA polymerases. The FDIET/C motif (using the single-letter code) occurring in this region was one of the mosthighly conserved sequences in yeast DNA polymerases IIand III and herpes viral family DNA polymerases, FDIE

Table 1. Spontaneous mutator phenotype of pol24 strainsSpontaneous mutation

Parent Marker rate (x 108) Ratiostrain gene POL2 pol24 pol2-4/POL2

CG379 adeS-J 17 ± 9.8 730 ± 63 43CG379 his7-2 0.20 ± 0.30 4.7 ± 1.4 24CG379 CANI 17 ± 1.4 86 ± 7.1 5AMY52-3D leu2-1 22 ± 1.9 400 ± 18 18AMY52-3D hom3-10 220 + 31 1900 ± 50 9AMY52-3D hisl-7 1.5 ± 0.9 46 ± 7.6 31

Spontaneous mutation was measured as reversion ofauxotrophs toprototrophy or, in the case of CANI, as forward mutation tocanavanine resistance. Numbers are the means (±SD) of threedeterminations using three independent isolates. pol24 genotypeswere confirmed by DNA sequencing.

V

POL 2

MDIET IV

Im

-E

0af0 -0

C 0

CoO

h-e

E00

CL< 5ZE,a C.

Biochemistry: Morrison et al.

9476 Biochemistry: Morrison et al.

r -e r--I-r

-LiX

He 7I 7

Li

--

-- Li= 0* - < ixk

1-i -1

'K".-

* ..... :1' -'

'II-'!

i..

'',.

..

Y I-.. ~,. :"

I

j

. - I

I'.!

F

-jo C,1

FIG. 4. Multiple amino acid sequence alignments ofthree regiproposed to comprise the 3' -- 5' exonuclease active site.Alignment proposed in this paper between E. coli DNA polymerI sequence containing exonuclease active site residues D355E357, other class A enzymes, and region of aphidicolin-sensiDNA polymerases amino-proximal to region IV. (B) Alignmessentially as proposed in refs. 12 and 13, between the E. coli Dpolymerase sequence containing D424, other class A polymeraand the right part of region IV of aphidicolin-sensitive DNA p

merases. (C) Alignment, essentially as proposed in refs. 4, 12,and 14, between the E. coli DNA polymerase sequence contaijY497 and D501, other class A polymerases, and region ofaphidiccsensitive DNA polymerases between regions IV and II. Astermark residues of the 3' -- 5' exonuclease active site of E. coli Dpolymerase I. Boxed are prominent identities and similarities sco

being the only tetrapeptide sequence absolutely conserved inthis set of polymerases. Similar motifs, similarly locatedrelative to region IV, were identified by computer alignmentin A. californica nuclear polyhedrosis virus, adenovirus, andvaccinia virus DNA polymerases (Fig. 1A).We realized that the yeast DNA polymerase II sequence

VmAFDiET strikingly resembled the sequence VfAFDtETof E. coli DNA polymerase I (lower-case letters representnonidentical residues), which contains the D355 and E357 3'-* 5' exonuclease active site residues (Fig. 1B). Moreover, afeature common to the aphidicolin-sensitive polymerasesaligned in Fig. 1A and the E. coli DNA polymerase I-relatedenzymes aligned in Fig. 1B is that the Asp and Glu corre-sponding to the E. coli D355 and E357 are the only invariantresidues. In comparison, the left part of region IV, in whichthese exonuclease active site residues were previouslythought to be conserved (11-13), does not contain an invari-ant Asp residue corresponding to critical (10) E. coli DNApolymerase I residue D355 (Fig. 1C). We proposed, then, thatthe FDIET sequence (residues 289-293) of yeast DNA poly-merase II, rather than any sequence in the left part of regionIV, corresponded to the E. coli DNA polymerase I sequencethat includes the 3' -- 5' exonuclease active site residues

D355 and E357.Mutation of the FDIET Sequence Motif ofDNA Polymerase

U. In experiments similar to those ofDerbyshire et al. (9), wealtered POL2, the gene determining DNA polymerase II, toinclude the code for the D290 -* A and E292 A mutations.

A yeast strain expressing the mutant allele, named poi24,was created by targeted integration of plasmid YIpJB1 (Fig.2). We partially purified DNA polymerase II in the singlepolypeptide form that retains the intrinsic 3' -- 5' exonu-

clease activity (21, 27-29). Following Mono S column chro-matography, the wild-type strain displayed the expected peakof polymerase activity coincident with a 3'-* 5' exonuclease

peak, assayed as single-stranded DNA nuclease (Fig. 3). Thepoi24 mutant yeast yielded an amount ofpolymerase activitysimilar to that of the wild-type strain, but no coincident peakof exonuclease activity was detected. A trace of nuclease,present in all fractions assayed and equivalent to 0.9%o of thepeak ofthe wild-type activity, limited estimation ofthe extentof the reduction in exonuclease to about 100-fold. Essentiallythe same profiles were obtained when 3' -+ 5' exonuclease

activity was measured by the more specific terminal mis-match excision assay (ref. 21; unpublished observations).Although we have not ruled out other formal possibilities,such as a structural disorganization of the mutant enzymecaused by the amino acid substitutions, the observed exo-nuclease deficiency is strikingly consistent with the assign-ment of active site residues predicted from the amino acidsequence alignments. In the case ofE. coliDNA polymeraseI, crystallographic analysis of the mutant protein showed noalteration in protein structure other than the expectedchanges at the mutation sites (9).

Spontaneous Mutator Phenotype. The presumed in vivo roleof 3' -- 5' exonucleases of eukaryotic DNA polymerases is to

ions edit out incorrectly incorporated nucleotides, and elimination(A) of this function is expected to result in a spontaneous mutatorrase phenotype. Consistent with this, the YIpJB1 integrant ofstrainanditivelent,)NALses,loly-,13,ningDlin-isks)NAring

at least 0.5 on the Dayhoff matrix. Horizontal lines separate aphid-icolin-sensitive DNA polymerases (above line) from E. coli DNApolymerase I and related enzymes (below line). Residues arescreened where their mutation has resulted in exonuclease-deficientDNA polymerase: residues 355, 357, 424, 497, and 501 ofE. coliDNApolymerase 1 (9, 10); residues 5 and 7 ofT7 polymerase (30); residues12, 14, and 66 of 4i29 polymerase (13); residues 290 and 292 of yeastDNA polymerase II (present work); residue 324 of T4 polymerase(ref. 14; exonuclease was determined for the double mutant ofresidues 191 and 324). See legend to Fig. 1 for abbreviations.

fin

.!

,..

E

:.

B

;

,. (t

:'. :,.

A. .: i'

C'

...

..

O.-:

Proc. Nad. Acad. Sci. USA 88 (1991)

Proc. Natl. Acad. Sci. USA 88 (1991) 9477

CG379 displayed about a 40-fold increase in spontaneousreversion of adeS-i (data not shown). Following growth onmedium containing 5-fluoroorotic acid to select for excision ofthe integrated plasmid, 83% of the derivatives retained themutator phenotype. A 0.55-kb region of the yeast chromo-somal DNA normally encoding the FDIET amino acid se-quence was amplified and sequenced. Yeast DNA amplifiedfrom four ofthe nonmutators had the DNA sequence encodingFDIET, whereas DNA from four ofthe mutator strains had thesequence encoding FAIAT, showing that the mutator pheno-type segregated with thepol24 genotype. The same procedureproduced similar results with strain AMY52-3D, where mu-tagenesis was measured as reversion of hisl-7 (data notshown). Spontaneous mutagenesis rates, measured at six lociin strains CG379 and AMY52-3D, were observed to increase5- to 43-fold in thepol24 derivatives (Table 1). poi24 mutants,however, displayed no significant defects in growth at 300C or370C or in sensitivity to UV or methyl methanesulfonate(unpublished observations), indicating that the DNA polymer-ase 11 3' -. 5' exonuclease has no essential role in cell growthor in the repair of DNA damage.Conserved 3' -- 5' Exonuclease Active Site. We have

presented evidence for the apparent conservation of the E.coli DNA polymerase 3' --* 5' exonuclease active site

residues D355 and E357 in a region of aphidicolin-sensitiveDNA polymerases different from that previously proposed(11-13). However, the results of our multiple amino acidsequence alignments concur with previous observations (4,12, 13) that E. coli DNA polymerase I residues D424, Y497,and D501 appear to be conserved in the right part of, anddistal to, region IV. The proposed alignments are shown inFig. 4. Five 3'-* 5' exonuclease active site residues of E. coliDNA polymerase I appear to be absolutely conserved in theaphidicolin-sensitive DNA polymerases-except for a DNApolymerases, which are discussed below. (We did not locateconserved residues corresponding to E. coli residue L361 orF473.) This argues that class B and class A DNA polymerasesare in fact related, as has been suggested (31).

Exonuclease Active Site Residues Are Not Identified in aDNAPolymerases. No example of the sequence DXE (where X isany amino acid), corresponding to E. coli DNA polymerase Iresidues 355-357, is conserved in alignments of the aminoportions ofhuman, yeast, and Drosophila aDNA polymerases(ref. 2; Hirose and Matsukage, personal communication).Strikingly, an Asp residue proposed to correspond to E. coliDNA polymerase I residue D424 is invariant in aphidicolin-sensitive DNA polymerases known to possess 3' -* 5' proof-

reading exonuclease activity yet occurs in none of the three aDNA polymerases aligned in Fig. 4B. (Human DNA polymer-ase a has a Glu in position 643, which might potentiallysubstitute for the Asp.)A previous proposal that some ofthese3' -* 5' exonuclease active site residues are conserved in

human and yeast a DNA polymerases was based on differentamino acid sequence alignments (13). Thus, in ref. 13, 3' + 5'exonuclease active site residues were assigned to the left andright parts of region IV ofthe human enzyme but to sequencesamino-proximal to region IV, as defined in refs. 2, 14, 26, and32, in the yeast enzyme. Furthermore, the yeast DNA poly-merase a sequence matched to the YX3D motifofE. coliDNApolymerase I residues 497-501 was given in ref. 13 as YQVCEbut should read YQVTCE (residues 720-725). Although a

sequence with the same spacing of aromatic and Asp residuesas the YX3D motif is conserved close to region II of a DNApolymerases (14)-residues 810-814 (FIVPD) of the yeastenzyme, residues 801-805 (YIVPD) ofthe human enzyme, andresidues 824-828 (YIVPD) of the Drosophila enzyme-thesesequences were not aligned by the BESTFIT program with the

YX3D motif conserved in other aphidicolin-sensitive poly-merases (unpublished observation). This corroborates an ear-lier observation that the region containing the YX3D motif,

referred to as region A, is not conserved in a DNA polymer-ases (3). It thus appears that none of the five proposedexonuclease active site residues can be identified in thecatalytic subunits of a DNA polymerases. This may explainwhy the catalytic subunits of yeast and human a DNApolymerases, purified from overproducing strains, are devoidof detectable 3' -- 5' proofreading exonuclease activity (ref.33; W. C. Copeland and T. S.-F. Wang, personal communi-cation).We thank Drs. T. Hirose and A. Matsukage for communicating the

unpublished amino acid sequence of Drosophila DNA polymerase aand an alignment with the human DNA polymerase a sequence. Wealso thank Dinh C. Nguyen for sequencing DNA amplified by thepolymerase chain reaction and Roel M. Schaaper and Kenneth R.Tindall for their critical reading of this manuscript.

1. Jung, G., Leavitt, M. C., Hsieh, J.-C. & Ito, J. (1987) Proc. Natl.Acad. Sci. USA 84, 8287-8291.

2. Wang, T. S.-F., Wong, S. W. & Korn, D. (1989) FASEBJ. 3, 14-21.3. Gibbs, J. S., Chiou, H. C., Bastow, K. F., Cheng, Y.-C. & Coen,

D. M. (1988) Proc. Natl. Acad. Sci. USA 85, 6672-6676.4. Hall, J. D., Wang, Y., Pierpont, J., Berlin, M. S., Rundlett, S. E.

& Wong, S. (1989) Nucleic Acids Res. 17, 9231-9244.5. Dorsky, D. I. & Crumpacker, C. S. (1990) J. Virol. 64, 1394-1397.6. Bernad, A., Lazaro, J. M., Salas, M. & Blanco, L. (1990) Proc.

Natl. Acad. Sci. USA 87, 4610-4614.7. Jung, G., Leavitt, M. C., Schultz, M. & Ito, J. (1990) Biochem.

Biophys. Res. Commun. 170, 1294-1300.8. Marcy, A. I., Hwang, C. B. C., Ruffner, K. L. & Coen, D. M.

(1990) J. Virol. 64, 5883-5890.9. Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L.,

Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240,199-201.

10. Derbyshire, V., Grindley, N. D. F. & Joyce, C. M. (1991) EMBOJ.10, 17-24.

11. Spicer, E. K., Rush, J., Fung, C., Reha-Krantz, L. J., Karam, J. D.& Konigsberg, W. H. (1988) J. Biol. Chem. 263, 7478-7486.

12. Leavitt, M. C. & Ito, J. (1989) Proc. Nail. Acad. Sci. USA 86,4465-4469.

13. Bernad, A., Blanco, L., Lazaro, J. M., Martin, G. & Salas, M.(1989) Cell 59, 219-228.

14. Reha-Krantz, L. J., Stocki, S., Nonay, R. L., Dimayuga, E., Goo-drich, L. D., Konigsberg, W. H. & Spicer, E. K. (1991) Proc. Natl.Acad. Sci. USA 88, 2417-2421.

15. Syvaoja, J., Suomensaari, S., Nishida, C., Goldsmith, J. S., Chui,G. S. J., Jain, S. & Linn, S. (1990) Proc. Natl. Acad. Sci. USA 87,6664-6668.

16. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) MethodsEnzymol. 154, 367-382.

17. Morrison, A., Araki, H., Clark, A. B., Hamatake, R. K. & Sugino,A. (1990) Cell 62, 1143-1151.

18. Rothstein, R. (1983) Methods Enzymol. 101, 202-211.19. Boeke, J. D., LaCroute, F. & Fink, G. R. (1984) Mol. Gen. Genet.

197, 345-346.20. von Borstel, R. C. (1978) Methods Cell Biol. 20, 1-24.21. Hamatake, R. K., Hasegawa, H., Clark, A. B., Bebenek, K.,

Kunkel, T. A. & Sugino, A. (1990) J. Biol. Chem. 265, 4072-4083.22. Sanjanwala, B. & Ganesan, A. T. (1989) Proc. Natl. Acad. Sci.

USA 86, 4421-4424.23. Ito, J. & Braithwaite, D. K. (1990) Nucleic Acids Res. 18, 6716.24. Gribskov, M. & Burgess, R. R. (1986) Nucleic Acids Res. 14,

6745-6763.25. Kouzarides, T., Bankier, A. T., Satchwell, S. C., Weston, K.,

Tomlinson, P. & Barrell, B. G. (1987) J. Virol. 61, 125-133.26. Boulet, A., Simon, M., Faye, G., Bauer, G. A. & Burgers, P. M. J.

(1989) EMBO J. 8, 1849-1854.27. Wintersberger, U. & Wintersberger, E. (1970) Eur. J. Biochem. 13,

11-19.28. Chang, L. M. S. (1977) J. Biol. Chem. 252, 1873-1880.29. Budd, M. E., Sitney, K. C. & Campbell, J. L. (1989) J. Biol. Chem.

264, 6557-6565.30. Patel, S. S., Wong, I. & Johnson, K. A. (1991) Biochemistry 30,

511-525.31. Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. (1990)

Protein Engineering 3, 461-467.32. Iwasaki, H., Ishino, Y., Toh, H., Nakata, A. & Shinagawa, H.

(1991) Mol. Gen. Genet. 226, 24-33.33. Brooke, R. G., Singhal, R., Hinkle, D. C. & Dumas, L. B. (1991)J.

Biol. Chem. 266, 3005-3015.

Biochemistry: Morrison et al.