THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 257. No. 4, Issue of February 25, pp. 1958-1964, 1982 Printed in U.S.A.

Nucleotide Sequence of the Escherichia coZipoZA Gene and Primary Structure of DNA Polymerase I*

(Received for publication, February 17, 1981, and in revised form, August 20, 1981)

Catherine M. Joyce, William S. KelleySg and Nigel D. F. Grindley From the Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, Connecticut 06510 and the $Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213

We report the nucleotide sequence of a 3.2 kilobase pair region of the Escherichia coli polA gene, compris- ing the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S . (1982) J. BioL Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our se- quence.

Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem Biophys. Res. Commun 37,982-989; Klenow, H., and Henningsen, I. (1970) Proc. Nutl. Acad Sei. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polAl and wild type alleles, we have identified the poZAl mutation as a change from Trp (TGG) to amber (TAG) at residue 342.

DNA polymerase I of Escherichia coli is a multifunctional single subunit enzyme. Since the discovery of this enzyme in 1956, a large body of research (reviewed in Ref. 1) has eluci- dated the enzymology of its three catalytic activities (polym- erization, 5’-3’ and 3’-5’ exonucleolytic digestion). Coordina- tion of the polymerization and 3’-5’ exonuclease activities allows error-free primed synthesis of DNA. Coordination of all three enzymatic activities results in nick translation in vitro, a model reaction for the enzyme’s presumed in vivo function in excision repair, and the removal of RNA primers from Okazaki fragments during discontinuous replication. In contrast to this detailed enzymological description, our phys- ical picture of the DNA polymerase I molecule is limited to the observation that the protein comprises two domains sep- arable by mild proteolysis (2, 3), the smaller NHn-terminal domain containing the 5 ‘ 3 exonuclease activity, while the larger COOH-terminal domain carries the polymerase and 3’- 5‘ exonuclease activities (4, 5). The lack of a more detailed physical description of the enzyme molecule can be attributed to technical difficulties resulting from both the large size of DNA polymerase I (about 100,000 daltons) and its relatively low abundance in cell extracts. The cloning of the DNA

* This work was supported by Grant GM-28550 from the National Institutes of Health. The costs of publication of this article were dekayed 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.

8 Recipient of Career Development Award FRA-198 from the American Cancer Society. Present address, Biogen, 50 Church Street, Cambridge, MA 02138.

polymerase I structural gene (poZA) onto phage X (6, 7) has circumvented one of these problems, making it relatively easy to prepare the polymerase in sufficient quantities for X-ray crystallography and protein chemistry. Moreover, the cloned polA gene serves as a convenient source of DNA for sequenc- ing. In this paper, we report the nucleotide sequence of a 3.2 kb’ region of the polA gene, and the amino acid sequence of the DNA polymerase 1 protein. The following paper (8) de- scribes protein chemical experiments that c o n f i i our se- quence.

EXPERIMENTAL PROCEDURES

Preparation of DNA-Phage NM852 (ApolA att+Nam7am53cl+) was kindly provided by Dr. N. E. Murray, University of Edinburgh. Phage particles were purified by two cycles of CsCl density gradient centrifugation. DNA was released by treatment of the phage with sodium dodecyl sulfate, and dialyzed against 10 m~ Tris-HC1, pH 8.0, 1 mM EDTA.

Plasmid DNA was prepared from cleared lysates of chloramphen- icol-amplified cultures, by CsCl density gradient centrifugation in the presence of ethidium bromide (9).

Restriction Endonuclease Mapping-Restriction enzymes were purchased from New England Biolabs (Beverly, MA) or Bethesda Research Laboratories. Hinff was a gift from Dr. M. D. Rosa, Yale University. DNA from phage NM852 was digested with HindIII, treated with alkaline phosphatase (Boehringer), and 5’ end labeled using [y:”P]ATP (New England Nuclear) and T4 polynucleotide kinase (P-L Biochemicals). After cleavage with Sac I, the relevant fragments were isolated by preparative agarose gel electrophoresis followed by electroelution (10). The labeled polA-containing frag- ments were mapped using the partial digestion technique of Smith and Birnstiel (11).

DNA Sequencing-The preparation of end-labeled restriction frag- ments for sequencing was essentially as described by Maxam and Gilbert (12). Plasmid DNA was digested with an appropriate restric- tion enzyme and end labeled as described above. Labeled fragments were separated by electrophoresis on polyacrylamide gels, and eluted from the crushed gel slice with high salt buffer. Singly labeled DNA fragments were obtained either by digestion with a second restriction enzyme or by electrophoretic strand separation.

Fragments were sequenced using the partial chemical degradation technique of Maxam and Gilbert (12). An A > C reaction was routinely used in addition to the G, A + G, C, and C + T reactions. The chemically cleaved products were examined on thin sequencing gels (13) containing 208,856, or 6% acrylamide. Sequence data were stored and analyzed using the computer programs developed by Staden (14).

RESULTS

Restriction Mapping-The ApolA phage constructed by Kelley et al. (6) carries the polA gene on a 5 kb HindIII fragment. We constructed a detailed restriction map of this region using the technique of Smith and Bimstiel (11). The polA-containing fragment was labeled at the HindIII ends and divided into two singly labeled fragments by cleavage at the unique Sac I site. From digestion of each fragment with

’ The abbreviations used are: kb, kilobase pair(s); bp, base pair(s).

1958

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Sequence of the E. coli polA gene 1959

12 different restriction enzymes under partial cleavage condi- tions, we were able to derive a restriction map that was sufficiently detailed to serve as a starting point for our DNA sequencing experiments. The data were checked by mapping each HindIII-Sac I fragment in the opposite direction, after labeling at the Sac I site. A restriction map derived in this manner should be reliable except in the immediate vicinity of the Hind111 and Sac I sites. Our DNA sequencing results showed this to be the case.

Construction of a Small polAl Plasmid-To facilitate the preparation of large quantities of DNA for sequencing, we decided to subclone the 5 kb HindIII fragment onto a small multicopy plasmid. Although the wild type polA gene cannot be stably maintained on a multicopy vector, the polAl amber mutant can be propagated in this way (6). We therefore obtained a plasmid (kindly provided by Dr. N. E. Murray) canying the 5 kb HindIII fragment from a polAl mutant in the vector pBR313 (15), and recloned this fragment into the smaller vector pNG16 (16). Fig. 1 shows a simplified restriction map of the resulting plasmid, pCJ1. It is about 8.5 kb in size, conveniently small for the preparation of DNA fragments for sequencing.

DNA Sequencing-Fig. 2B shows the strategy used for sequencing the poZ.4 gene and surrounding regions, and the extent of sequences determined. More than 97% of the se- quence was determined on both DNA strands and overlapping data were obtained for all restriction sites. Fig. 3 gives the nucleotide sequence of the region covered by Fig. 2; it com-

Hlnd 111

Sac I

pCJ I 8.5 kb

ApR Tcs

FIG. 1. Simplified restriction map of the plasmid pCJ1. The cloned HindIII fragment is represented by the heavier line. The boxed region contains the polA coding sequence and is shown in more detail in Fig. 2. Transcription of the polA gene starts near the Bgl I 1 site (17) and proceeds in the direction shown by the arrow.

prises the DNA polymerase I coding region plus about 400 bp of untranslated sequence.

Previous studies on ApolA-lacZ fusions (17) indicated that the polA promoter is close to the Bgl TI site and that the direction of transcription is as shown in Fig. 1. Consistent with these results, we located the start of the coding region 100 bp downstream from the BgZ I1 site by scanning our translated DNA sequence for the previously determined NH2-terminal amino acid sequence (5). As shown in Table I, there is total agreement between our predicted sequence and the experi- mental results through six cycles of Edman degradation.

Having identified the start of the coding sequence, we observed an open translational reading frame extending for a total of 928 codons from the initiator ATG, interrupted only by a single amber codon at residue 342. To c o n f i i that this amber codon was, as we suspected, the site of the poLAl mutation, we sequenced the corresponding Xho I fragment from phage NM852 (ApoZA+). Comparison of the two se- quences showed that the polAl mutation corresponds to a change from TGG (Trp) to TAG (amber) at residue 342. We have sequenced all the polA-containing Xho I fragments from NM852, a total of 830 bp (Fig. 2C), and have found the wild type sequence over this region to be identical with that derived from pCJl (polAI), aside from the single amber codon. This observation suggests that, although derived by heavy nitro- soguanidine mutagenesis (18), polAl is a single point muta- tion, consistent with previous genetic data (19). (A further argument against gross chromosomal rearrangements in the polA1 fragment cloned on pCJl is provided by the good correspondence between the experimentally determined re- striction map (from phage NM852) and that derived from the DNA sequence of pCJ1.) On the assumption that there are no further differences between the wild type and mutant se- quences, we have derived the amino acid sequence of DNA polymerase I shown in Fig. 3.

DISCUSSION

We have determined the DNA sequence of the coding region of the polA gene and from this we have deduced the amino acid sequence of DNA polymerase I. Since almost the entire sequence was obtained by sequencing both DNA strands, we are confident that it is correct. We have also examined our sequence to see whether it is consistent with data obtained for the DNA polymerase I protein molecule.

Comparison with Protein Chemical Data-As shown in Table I, our sequence agrees with the previously determined NH2-terminal sequence (5) through six cycles of Edman deg- radation. The divergence of the two sequences after the sixth

A Small fragment Large fragment

5'-3' exonuclease pol *I Polymerase 3-5' exonuclease

I ......................................... 704

-300 I 500 1000 IS00 2000 2500 M O O bp I I I : I

P

Ava II Hoe Ill Hid I SalrSA I Hpa II

C I- t"-+ Xho I

FIG. 2. The E. coli polA gene. A, organization of the gene. The sequence is numbered from the translational ini- tiation point. The boxed area comprises the coding region which is divided into regions corresponding to the large (ma) and small (a) proteolytic fragments. The vertical arrow marks the position of the pool1 mutation at nucleotide 1025. B, strategy used to determine the nucleo- tide sequence from the plasmid pCJ1. The vertical bars indicate the restriction sites for the enzymes used in sequencing. The horizontal arrows show the extent of sequence determined from each 5"end labeled fragment. C, extent of nucleotide sequence determined from phage NM852 (ApolA').

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1960 Sequence of the E . coli polA gene -300 -250

ATCCITAAGG AGAAAAATAA TTCATATCTA TCCACATTAG AAAAAATCCC ATTATCTCAA TTAITAGOGA V X A T I T A T T T l T M C T G C A TGAAAAACAA

-200 AGACAAACAT C A m T A A AAAGCATGAT AATAAATTAA AAGCGATGTA AATAATITAT GCACAAAGIT ATCCACATGA C G A m C C G A GCGATCCAGA

-150

-100 -50 AGATCTACAA A A G A T l l T A CGAAAAGCGG TGAAAAACTC ATGTI lTCAT CCTGTCIY;TG GCATCCTITA CCCATAATCT GATAAACAGG CACGGACATT

Arc GIT CAG ATC ccc CAA AAT CCA CTT AX CTT GTA GAT GOT TCA TCT TAT CTT TAT CGC GCA TAT CAC GCG m ccc CCG n~ ACT AAC 30 60 90

MET VAL GLN ILE PRO GLN ASN PRO LEU ILE LEU VAL ASP GLY SER SER 'NR LEU TKR ARG ALA TYR HIS ALA PHE PRO PRO LEU I R R ASH 1

AGC GCA W C GAG CCC ACC GGT GCG ATG TAT GGT GTC CTC AAC ATG CTG CGC ACT CTG ATC ATG CA.4 TAT AAA CCG ACG CAT GCA GCG GTG SER ALA GLY GLU PRO TEP, GLY ALA MET TYR GLY VAL LEU ASN MET LEU ARC SER LEU ILE NET G L N TYR LYS PRO I R R HIS ALA ALA VAL 31

120 150 180

G T C TIT GAC GCC AAG GGA AAA ACC TIT COT GAT GAA CTG TIT GAA CAT TAC AAA TCA CAT CGC CCG CCA ATG CCG GAC GAT CTG CGT GCA VAL PHE ASP ALA LYS GLY LYS I R R PEE ARC ASP GLU LEU PHE GLU HIS TYR LYS SER HIS ARG PRO PRO IET PRO ASP ASP LEU ARC ALA 6 1

210 240 270

CAA ATC GAA CCC TTG CAC GCG ATG G T T AAA GCG ATG GGA CTG CCG CTG CTG GCG G I T TCT GGC GTA G M GCG GAC GAC G n ATC GGT ACT 300 330 360

GLPl ILE GLU PRO LEU BIS ALA MET VAL LYS ALA MET GLY LEU PRO LEU LEU ALA VAL SER GLY VAL GLU ALA ASP ASP VAL ILE GLY TKR 91

CTG GCG CGC GAA GCC GAA AAA GCC GGG CGT CCG GTG CTG A X AGC ACT GGC GAT A M GAT ATG GCG CAG CTG GTG ACG CCA AAT A T I ACG 3 90 420 450

LEU ALA ARC GLU ALA GLU LYS ALA GLY ARC PRO VAL LEU ILE SER l R R GLY ASP LYS ASP !.!ET ALA G L N LEU VAL TER PRO ASN ILE TKR 121

CTT ATC AAT ACC ATG ACG AAT ACC ATC CTC GGA CCG GAA GAG GTG GTG AAT AAG TAC GGC GTG CCG CCA GAA CTG ATC ATC GAT TTC CTG 480 51 0 540

LEU ILE ASN TRR MET TER ASN m R ILE LEU GLY PRO GLU GLU VAL VAL ASN LYS ' N R GLY VAL PRO PRO GLU LEU ILE ILE ASP PEE LEU 151

GCG CTG ATG GOT GAC TCC TCT GAT AAC ATT CCT GGC GTA CCG GGC GTC GGT GAA AAA ACC GCG CAG GCA TTG CTG CAA GGT CTT GGC GGA 570 600 630

ALA LEU MET GLY ASP SER SER ASP ASN ILE PRO CLY VAL. PRO GLY VAL GLY GLU LYS I R R ALA GLN ALA LEU LEU CLN GLY LEU GLY GLY 1 81

I X G GAT ACG CTG TAT GCC GAG CCA GAA A M ATT GCT GGG TTG AGC TIC CGT GGC GCG A M ACA ATG GCA CCG AAG CTC GAG CAA AAC A M 660 6 90 720

LEU ASP THR LEU TYR ALA GLU PRO GLU LYS ILE ALA GLY LEU SER PHE ARC GLY ALA LYS I R R MET ALA ALA LYS LEU GLU GLN ASN LYS 211

GAA G I T GCT TAT CTC TCA TAC CAG CTG GCG ACG A T I A M ACC GAC G T T GAA CTG GAG CTG ACC TGT GAA CAA CTG GAA GTG CAG CAA CCG 750 7 80 810

GLU VAL ALA TYR LEU SER TYR G L N LEU ALA I R R ILE LYS I R R ASP VAL GLU LEU GLU LEU IRP. CYS GLU GLN LEU GLU VAL G L N GLN PRO 241

GCA GCG GAA GAG TTG I T G GGG CTG TTC AAA AAG T A T GAG TTC AAA COC TGG ACI' GCT GAT GTC GAA GCG GGC A M TGG TTA CAG GCC A M 870 900

ALA ALA GLU CLU LEU LEU GLY LEU PHE LYS LYS TYR GLU PEE LYS ARC TRP I R R ALA ASP VAL GLU ALA GLY LYS TRP LEU G L N ALA LYS 271

GCG GCA AAA CCA GCC GCG AAG CCA CAG GAA ACC ACT G I T GCA GAC G M GCA CCA G A A GTG ACG GCA ACG GTG A T T TCT TAT GAC AAC TAC 960 9 90

GLY ALA LYS PRO ALA ALA LYS PRO G L N GLU IRE SER VAL ALA ASP GLU ALA PRO GLU VAL THR ALA IRR VAL ILE SER TYR ASP ASN TYR 301

GTC ACC ATC CTT GAT GAA GAA ACA CTG A M GCG TGG ATT GCG AAG CTG GAA A M GCG CCG GTA TIT GCA TIT GAT ACC GAA ACC GAC AGC 1020 TAG (polAl) 1050 1080

VAL THR ILE LEU ASP GLU GLU I R R LEU LYS ALA TRP ILE ALA LYS LEU GLU LYS ALA PRO VAL PHE ALA PIE ASP TER GLU I R R ASP SER 331

CTT GAT AAC ATC TCT GCT AAC CTG G X GGG CTT TCT m GCT A X GAG CCA GGC GTA GCG GCA T A T A T I CCG G T T GCT CAT GAT TAT CTT LEU ASP ASN ILE SER ALA ASN LEU VAL GLY LEU SER PHE ALA ILE GLU PRO GLY VAL ALA ALA TYR ILE PRO VAL ALA BIS ASP TYR LEU 361

GAT GCG CCC GAT CAA ATC TCT CGC GAG CGT GCA CTC GAG TTG CTA AAA CCG CTG CTG GAA GAT GAA AAG GCG CTG AAG GTC GGG CAA AAC ASP ALA PRO ASP GLN ILE SER ARC GLU ARC ALA LEU GLU LEU LEU LYS PRO LEU LEU GLU ASP GLU LYS ALA LmT LYS VAL GLY GLN ASN 391

CTG AAA TAC GAT CGC GGT A l T CTG GCG AAC TAC GGC ATT GAA CTG CGT GGG ATT GCG T I T GAT ACC ATG CTG GAG TCC TAC A T I CTC AAT 1290 1320 1350

LEU LYS TYR ASP ARC GLY ILE LEU ALA ASN ' N R GLY ILE GLU LEU ARC GLY ILE ALA PEE ASP TER MET LEU GLU SER TYR ILE LEU ASN 421

AGC G T T GCC GGC CGT CAC GAT ATG GAC AGC CTC GCG FAA COT TGG TTG AAG CAC A M ACC ATC ACT TIT GAA GAG A 1 T GCT GGT A M GGC 13 80 1410 1440

SEE VAL ALA GLY ARG IiIS ASP MET ASP SER L E U ALA GLU ARC TRP LEU LYS 51s LYS TIiR ILE IRR PHE GLU GLU ILE ALA GLY LYS GLY 451

AAA AAT CAA CTG ACC TIT AAC CAG ATT GCC CTC GAA GAA GCC GGA CGT TAC GCC GCC GAA GAT GCA GAT G T C ACC TT.3 CAG TTG CAT CTG 1470 1500 1530

LYS ASN CUI LEU TW. PHE ASN GLN ILE ALA LEU GLU GLU ALA GLY ARC TYP. ALA ALA GLU ASP ALA ASP VAL I R R LEU GLN LEU 51s LEU 481

840

930

e..

1110 1140 1170

1200 1230 1260

FIG. 3. Nucleotide sequence of the E. coli poZA gene and amino acid sequence of DNA polymerase I. The DNA sequence is numbered above each line, designating the translational start as +I. The protein sequence is numbered below each line. * * * at residue 324 indicates the NH2 terminus of the large proteolytic fragment. The poZAl mutation at nucleotide 1025 is shown.

residue is probably not surprising since Jacobsen et al. (5) sequence Val-Ile-Met at the NH2 terminus of the larger (PO- tentatively assigned the final two residues on the basis of one lymerase proficient) fragment. However, we were unable to further cycle of Edman degradation in which both leucine and locate this tripeptide on our protein sequence. Brown et al. aspartate were detected. (8) therefore redetermined the NH2-terminal sequence of pu-

Mild proteolysis of DNA polymerase I removes the NH2- rified large fragment using an automatic sequencer. The re- terminal one-third of the molecule, producing two enzymati- sults (see following paper) indicated that the third amino acid cally active fragments (2, 3). Jacobsen et al. (5) identified a determined by Jacobsen et al. (5) was incorrect and allowed

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Sequence of the E . coli polA gene 1961

AM ATG TGG CCG GAT CTG CAA AAA CAC AM GGG CCG TTG AAC GTC TIT GAG M T ATC GAA ATG CCG CTG GTG CCG G T G CIT TCA CGC ATT 1560 1590 1620

LYS MET TRP PRO ASP LEU GLN LYS EIS LYS GLY PUO LEU ASN VAL PHE GLU ASN ILE GLU MET PRO LEU VAL PRO VAL LEU SER ARC ILE 511

G M CGT M C GGT GTG AAG ATC GAT CCG A M GTG CTG CAC AAT CAT TCT GAA GAG CTC ACC CIT CGT CTG GCT GAG CTG G M AAG A M GCG 1650 1680 1710

GLU ARC ASN GLY VAL LYS ILE ASP PRO LYS VAL LEU HIS ASN EIS SEI( GLU GLU Lrm TER LEU ARG LEU ALA GLU LEU GLU LYS LYS ALA 541

CAT G M A l T GCA GGT GAG G M TlT M C CIT TCT TCC ACC AAG CAG TTA C M ACC A l T CTC TIT GAA A M CAG GGC A l T AM CCG CTG AAG 17 40 1770 1800

HIS GLU ILE ALA GLY GLU GLU PEE ASN LEU SER SER TER LYS GLN LEU GLN TEBR ILE LEU PHE GLU LYS GLN GLY ILE LYS PRO LEU LYS 571

AAA ACG CCG GGT GGC GCG CCG TCA ACG TCG GAA GAG GTA CTG G M GAA CTG GCG CTG GAC TAT CCG TTG CCA AM G T G ATT CTG GAG TAT 1830 1860 1890

LYS THR PRO GLY GLY ALA PRO SER TER SER GLU GLU VAL LEU GLU GLU LEU ALA LEU ASP TYR PRO LEU PRO LYS VAL ILE LEU GLU TYR 6 0 1

COT GGT CTG GCG M G CTG A M TCG ACC TAC ACC GAC AAG CTG CCG CTG ATF ATC AAC CCG AM ACC GGG CGT GTG CAT ACC TCT TAT CAC 1920 1950 1980

ARC GLY LEU ALA LYS LEU LYS SER l I l R ' l Y R TER ASP LYS LEU PRO LEU MET ILE ASN PRO LYS TER GLY ARC VAL EIS TER SER TYR EIS 631

CAG GCA GTA ACT GCA ACG GGA COT TTA TCG TCA ACC GAT CCT M C CTG CAA M C ATT CCG GTG CGT M C G M G M GOT CGT CGT ATC CGC 2010 2040 2070

GLN ALA VAL TBR ALA TER GLY ARC LEU SER SER TER ASP PRO ASN LEU GLN ASN ILE PRO VAL ARC ASN GLU GLU GLY ARC ARC ILE ARG 661

CAG GCG TIT ATT GCG CCA GAG GAT TAT GTG ATT G l L TCA GCG GAC TAC TCG CAG ATT G M CTG CGC A l T ATG GCG CAT CIT T C G CGT GAC 2100 2130 2160

GLN ALA PIE ILE ALA PRO GLU A S P TYR VAL I I E VAL SER ALA A S P TYR S E R GLN ILE GLU LEU ARG ILE MET ALA EIS LEU SER ARC ASP 691

AM GGC l T G CTG ACC GCA TIT GCC G M GGA AAA GAT A X CAC COG GCA ACG GCG GCA G M GTG m GGT TI0 CCA CTG G M ACC GTC ACC 2190 2220 2250

LYS GLY LEU LEU TBR ALA PIE ALA GLU GLY LYS A S P ILE HIS ARC ALA l l i R ALA ALA GLU VAL PIE GLY LEU PRO LEU GLU 188 VAL 'IER 7 2 1

AGC GAG CAA CGC CGT AGC GCG AM GCG A K M C TlT GGT CTG ATT TAT GGC ATG ACT GCT TTC GGT CTG GCG CGG CAA TTC M C A T T CCA 2280 2310 2340

SER GLU GLN ARC ARC SER ALA LYS ALA ILE ASN PBE GLY L m ILE TYR GLY MET SER ALA PBE GLY LFXJ ALA ARC GLN LEU ASN ILE PRO 751

CGT A M G M GCG CAG M G TAC ATG GAC CIT TAC TIT G M CGC TAC CCT GGC GTG CTG GAG TAT ATG GAA CGC ACC CGT GCT CAG GCG A M 2370 2400 2430

Ap.G LYS GLU ALA GLN LYS TYR MET A S P LEU TXR PHE GLU ARC l T R PRO GLY VAL LEU GLU TYR MET GLU ARC TER ARC ALA GLN ALA LYS 781

GAG CAG GGC TAC G l T G M ACG CTG GAC GGA CGC CGT ( X G TAT CTG CCG GAT ATC A M TCC AGC AAT GGT GCT CGT CGT GCA GCG GCT G M 2460 2490 2520

GLU GLH GLY TYR VAL GLU E R LEU ASP GLY ARC ARG LEU TYR LEU PRO ASP ILE LYS SER SER ASN GLY ALA ARC ARG ALA ALA ALA GLU 811

CGT GCA GCC A l T M C GCG CCA ATG CAG GGA ACC GCC GCC GAC ATT A X AAA COG GCG A T F A l T GCC G I T GAT GCG TGG T T A CAG GCr GAG 2550 2580 2610

ARC ALA ALA ILE ASN ALA PRO MET G W GLY TRR ALA ALA ASP ILE ILE LYS ARC ALA MET ILE ALA VAL ASP ALA TRF' LEU G L N ALA GLU 841

CAA CCG CGT GTA CGT ATG ATC ATG CAG GTA CAC GAT G M CTG GTA TlT GAA G T T €AT AAA GAT GAT G T T GAT GCC GTC GCG AAG CAG ATT 2640 2670 2700

871

CAT CAA CTG AT0 GAA M C TGT ACC CGT CTG GAT G T G CCG TTG CTG GTG G M GTG GGG ACT GGC G M AAC TGG GAT CAG GCG CAC TAA GAT 2730 2760 27 90

111s GLPl LEU MET GLU ASN CYS TER ARG LEU ASP VAL PRO LEU LEU VAL GLU VAL GLY SER GLY GLU ASN TRP ASP GLN ALA HIS END 901

GLN PRO ARC VAL ARC MET ILE MET GLN VAL EIS ASP GLU LEU VAL PHE GLU VAL nIs LYS ASP ASP VAL ASP ALA VAL ALA LYS CLN ILE

TCGCCTGAAC ATGCCTITIT TCGTAAGTM CCAACATAAG CTGTCACGTT lTGTGATGGC TATTAGAMT TCCTATGCM CMCTGAAAA AAMTTACM 2840 2890

FIG. 3

us to position the start of the large fragment at residue 324. Nucleotide sequence data spanning this region is shown in Fig. 4.

A common error in DNA sequencing is the insertion or omission of a single nucleotide, resulting in a change in the reading frame of a translated gene product and probable misassignment of the COOH terminus of the protein. In the following paper, Brown et al. (8) present several lines of evidence supporting our identification of the COOH terminus of the DNA polymerase I molecule. The most direct evidence is the sequential release of histidine, alanine, and glutamine by carboxypeptidase digestion of DNA polymerase I or its large proteolytic fragment. Confirmatory data is provided by the isolation of the predicted COOH-terminal tryptic peptide, and by chemical cleavage experiments which position a cys- teine residue extremely close to the COOH terminus.

The amino acid compositions and molecular weights that we predict for DNA polymerase I and its two proteolytic fragments (Table 11) are in good agreement with experimen- tally determined values (5, 8, 20, 21). The single divergence between our data and earlier work is in the number of cys- teines. Previous work indicated that a molecule of the polym- erase contains three half-cystine residues, two of which are

involved in disulfide bond formation (20), and that all three half-cystines are present in the large proteolytic fragment (21). By contrast, our DNA sequence shows only two cysteine codons, consistent with the composition data of Brown et al. (8). The actual location of the cysteines, at positions 262 (small fragment) and 907 (large fragment), is confirmed by chemical cleavage experiments described in the following pa-

We should point out that the protein chemical data reported by the Kornberg and Klenow (1, 5) groups were obtained using DNA polymerase I from E. coli B, whereas the cloned polA gene which we have sequenced, and which Brown et al. (8) have used to prepare homogeneous polymerase, was de- rived from E. coli K12. However, we do not find this a convincing reason for the discrepancies discussed above, since our preliminary DNA sequence studies on the resAl and re& mutant alleles ofpoll , which were derived from E. coli B (22), have revealed no changes from the K12 sequence.'

The polsll Mutation-By comparing the p o l l 1 and p o l l +

sequences we have identified the pol l1 mutation as a change from Trp (TGG) to amber (TAG) at codon 342. This site is

per (8).

* C. M. Joyce, unpublished observations.

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1962 Sequence of the E. coli polA gene

just within the NH2-terminal region of the large proteolytic fragment (residue 324), suggesting that, in the absence of degradation, thepolAl amber fragment would be only slightly larger than the small proteolytic fragment. The identification in a polAl cell extract of a polypeptide indistinguishable in size and enzymatic activity from the small proteolytic frag- ment (4) is consistent with our assignment of the mutation site, and suggests that the polAl amber fragment is indeed relatively resistant to proteolysis.

TABLE I NHderminal Sequence of Whole Polymerase Predicted from DNA sequence: Met-Val-Gln-Ile-Pro-Gln-Asn-Pro-Leu-Ile

Experimental (5): Met-Val-Glx-Ile-Pro-GIx-Leu-Asx

;;"

Ile

Val (324)

Thr

- c fhr (321) A

FIG. 4. Nucleotide sequence data corresponding to the M I z - terminal region of the large proteolytic fragment. A restriction fragment labeled at the Hae I11 site at position 896 was chemically cleaved, as described by Maxam and Gilbert (12). The products were fractionated on an 8% polyacrylamide gel under denaturing condi- tions. From left to right, the slots correspond to A > C, G, A + G, C + T. and C specific reactions. The written sequence corresponds to positions 961-990 on Fig. 3.

Codon Usage-Table I11 details the codon frequencies in the E. coli polA gene. The observed distribution is similar to that found for other sequenced E. coli genes (tabulated in Ref. 23), and supports the notion that E. coli genes have characteristic codon preferences, distinct from phage- and transposon-encoded genes that are translated in E. coli (23, discussed in Ref. 24). Examples of the biased codon distribu- tion are the preference for CUG (Leu) and CGY (Arg) and the avoidance of AUA (Ile). As discussed by Post et al. (25),

TABLE I1 Amino Acid Composition of DNA Polymerase I and its Proteolytic

Sub-fragments Whole Large Small

polymerase" fragment fragmenth

ASP 51 35 16 Asn 32 23 9 Thr 50 29 21 Ser 39 27 12 Glu 80 54 26 Gln 39 25 14 Pro 50 27 23 GlY 57 33 24 Ala 99 62 37 CYS 2 1 1 Val 57 35 22 Met 25 14 11 Ile 53 39 14 Leu 1 0 6 69 37 TYr 32 21 11 Phe 24 16 8 His 21 16 5 LYS 59 38 21 Trp 7 5 2 Arg 45 36 9

Total 928 605 323

Molecular Weight 103,116 6 8 , 0 6 4 34,052 a Amino acid composition predicted from DNA sequence.

Values are calculated assuming that proteolysis results in a single cut between residues 323 and 324, with no subsequent degradation of the small fragment.

TABLE 111 Codon usage in the E. colipolA gene

Second base

U

16

8

4

15

Phe

U

LRI

14

10

1

62

C Leu

29

I l e 24

n A

Met 25

13

12

10

22

G V a l

C

10

4

7

5

Ser

3

4

14

29

Pro

6

29

2

13

Thr

13

16

23

47

Ala

Hi s 11

10

17

22 GI n

Asn 10

22

42

17 LYS

ASP 34

17

57

73 GI u

G

U 2

0 C

End 0 A

T r P 7 G

CYS

28

14

U

C

G 3

A AnJ

4

A 0

C 9

U

G

Ser

Arg

18 U

C 19

9 A

I 1 G

G ~ Y

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Sequence of the E . coli polA gene 1963

these biases correspond to use of the most abundant tRNA species. Further evidence that these codon preferences have biological significance, and are not merely a consequence of nonrandom nearest neighbor relationships in the E. coli ge- nome, is provided by the absence of the same biases in the two out of phase reading frames for thepolA region. Moreover, if future sequencing studies confirm our belief that this pattern of codon usage is typical of E. coli genes, an examination of codon distribution could provide a useful additional criterion for distinguishing between plausible and implausible reading frames within a DNA sequence.

A more detailed comparison of Table I11 with previously determined codon frequencies suggests that, although the polA gene shows the same preferences as other E. coli genes, the bias is less extreme than in genes for very abundant proteins (ribosomal proteins, Ref. 25, and the outer membrane lipoprotein, Ref. 24). It is tempting to speculate that the level of about 400 molecules/cell of DNA polymerase I (1) can be maintained without needing to optimize use of the E. coli translational machinery.

FZanking Sequences of the polA Gene-Fig. 3 shows ap- proximately 300 bp upstream and 100 bp downstream of the polA coding sequence. It is reasonable to suppose that these regions contain the sequences that control initiation and ter- mination of transcription of the polA gene.

Sequencing and biochemical studies of promoters recog- nized by E. coli RNA polymerase (reviewed in Refs. 26 and 27) indicate that they contain a highly conserved region about 10 bp upstream of the site of transcription initiation (the Pribnow box, prototypically TATAATPu), and a somewhat less well conserved “polymerase binding site” about 35 bp upstream (the -35 region). Examination of the DNA sequence preceding the polA coding region reveals many sites having some homology with the prototype bacterial promoter se- quence. The abundance of sites is probably a consequence of the high A T content of this region (about 70% between posi- tions -50 and -300 relative to the translational start). On the basis of sequence homology, we feel that the most plausible promoter sequence for the polA gene involves one of the Pribnow boxes at -28 to -22 (CATAATC) or -150 to -144 (AATAATT) (Fig. 3). However, in neither case is there con- vincing homology at the -35 region.

Our inability to locate the polA promoter merely by se- quence comparison may not be surprising. The majority of the promoter sequences used in deriving a consensus sequence belong to phage genes, stable RNA genes or genes for abun- dant bacterial proteins. Many of these genes are subject to additional forms of regulation. By contrast, polA is expressed at a relatively low level (about 400 molecules/cell, Ref. 1) and does not appear to be regulated (17). The only other low level, constitutively expressed bacterial gene whose promoter has been sequenced (ZacI, Ref. 28) also shows poor homology with the consensus sequence. However, the lacI promoter may not be a suitable model for polA, since the level of expression of lacI is about 30-fold lower (17, 28).

An additional factor in the low level of expression of the polA gene may be poor initiation of translation of the polA transcript. Potential base pairing with the 3‘-end of 16 S ribosomal RNA in the initiation complex (29) is limited to the 3-base sequence GGA (-7 to -5, Fig. 3).

On the E. coli genetic map (30) the ribosomal RNA operon, r r n 4 , immediately precedes poM. We have determined the DNA sequence upstream from the polA gene as far as the Hind111 site used in constructing the polAl clone (about N o bp to the left of the region shown in Fig. 2). We have found no sequences corresponding to ribosomal RNA sequences. At present, we do not know whether this region constitutes a

truly “silent” portion of the E. coli genome. We have identified the next gene downstream from polA as

the gene for a small RNA (spot 42) characterized and se- quenced by Sahagan and Dahlberg (31). Transcription of this RNA is initiated about 150 bp beyond the end of the polA coding sequence, suggesting that the polA transcript must terminate within the last 100 nucleotides shown in Fig. 3. Since this region contains no obvious dyad symmetries anal- ogous to previously characterized p-independent terminators (26), we expect that transcription termination may be p-de- pendent. Further experiments are in progress to locate the precise sequences involved in initiation and termination of the polA transcript, and thus to define the boundaries of thepolA gene.

Evolution of thepolA Gene-The fact that the DNA polym- erase I molecule contains two separable domains, each having DNA-binding and nucleolytic activities, together with the approximate 2:1 ratio of the sizes of the large and small proteolytic fragments, suggests that the polA gene may have resulted from the multiplication (probably triplication) of the gene for a primitive DNA-binding protein. Even after subse- quent sequence divergence, such an event might be expected to leave traces of homology between domains when examined at the DNA sequence level. Computer analysis of our sequence has revealed no evidence for such homology. The secondary structure prediction of Brown et al. (8) is also at odds with a triplicated structure for DNA polymerase I, since similar features of super secondary structure do not appear at regular intervals throughout the protein. We therefore believe that the polA gene did not arise from a gene multiplication of the type discussed above, and feel that similarities between active sites on the enzyme may only be apparent at the tertiary structure level.

Acknowledgments-We are grateful to Michael A. Shanblatt for invaluable help with computer programming.

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C M Joyce, W S Kelley and N D GrindleyDNA polymerase I.

Nucleotide sequence of the Escherichia coli polA gene and primary structure of

1982, 257:1958-1964.J. Biol. Chem. 

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