JOURNAL OF BACTERIOLOGY, Apr. 1989, p. 2101-2109 Vol. 171, No. 40021-9193/89/042101-09$02.00/0Copyright X3 1989, American Society for Microbiology

Suppression of a Frameshift Mutation in the recE Gene ofEscherichia coli K-12 Occurs by Gene Fusion

CHARLES C. CHU, ANN TEMPLIN, AND ALVIN J. CLARK*

Department of Moleclular Biology, University of California, Berkeley, California 94720

Received 1 August 1988/Accepted 2 January 1989

The nucleotide sequences of a small gene, racC, and the adjacent N-terminal half of the wild-type recE geneare presented. A frameshift mutation, recE939, inactivating recE and preventing synthesis of the active recEenzyme, exonuclease VIII, was identified. The endpoints of five deletion mutations suppressing recE939 weresequenced. All five delete the frameshift site. Two are intra-recE deletions and fuse the N- and C-terminalportions of recE in frame. Three of the deletions remove the entire N-terminal portion of recE, fusing theC-terminal portion to N-terminal portions of racC in frame. These data indicate that about 70% of theN-terminal half of recE is not required to encode a hypothesized protein domain with exonuclease VIII activity.

Escherichia coli K-12 strains undergo homologous geneticrecombination via the RecBC, RecE, and RecF pathways(14). In wild-type E. coli, RecBC pathways (for justificationof the plural used here, see reference 14) account for about99% of conjugational recombinants (13, 32). recB and recCmutant strains, in which RecBC pathways do not operate,can be restored to near normal levels of conjugationalrecombinant production by two types of suppressor muta-tions: sbcA and sbcB (44). RecF pahways lead to recombi-nants when sbcB is the mutation, and RecE pathways do thesame when sbcA is the mutation. sbcB mutations reduceDNA exonuclease I (Exol) activity (28, 30); sbcA mutationsallow expression of the recE gene (7).recE encodes DNA ExoVIII, a 140-kilodalton (kDa) ATP-

independent exonuclease, which processively digests duplexDNA, producing 3' tails that are presumably utilized inrecombination (23, 24, 29). recE resides on a defectiveprophage known as Rac (33). In this paper we present thesequence of the N-terminal half of recE. We show that acopy of recE, previously cloned from sbcA+ strain KL16(47), contains a frameshift mutation preventing expression ofenzyme activity, and we discuss the origin of the mutation.Finally, we describe the nature of deletions that suppress theframeshift mutation.

MATERIALS AND METHODSBacterial strains. All bacteria used in this work were

derivatives of E. coli K-12. For genetic nomenclature, theconventions of Demerec et al. (16) and Bachmann (6) arefollowed.

Plasmids and bacteriophages. Plasmids constructed in thiswork are listed in Table 1. Plasmids pRAC1, pRAC3,pRAC4, pRAC5, pRAC7, pRAC10, pRAC31, and pRAC36and the strains containing them were described by Willis etal. (47). pRAC2 was an independent clone isolated fromsbcA+ recE+ strain KL16 in the same experiment whichproduced pRAC1 (D. K. Willis, Ph.D. dissertation, Univer-sity of California, Berkeley, 1982). Besides a 7.65-kilobase(kb) HindIII fragment, similar in size and restriction patternto that in pRAC1, pRAC2 contains additional unmapped anduncharacterized HindIII fragments (Willis, Ph.D. disserta-

* Corresponding author.t Present address: Laboratory of Immunology, National Institute

of Allergy and Infectious Diseases, Bethesda, MD 20892.

tion). The construction of pSKM1 and plasmids carryingsbc::TnS alleles will be described elsewhere (S. K. Mahajanet al., submitted for publication; C. Chu and A. J. Clark,manuscript in preparation).

Plasmids pJC758, pJC843, pJC844, pJC845, pJC846, andpJC847 were constructed by EcoRI restriction enzyme di-gestion of DNA from the source plasmid DNA (Table 1),followed by treatment with ligase. The DNA mixtures wereused to transform strain AB1157 to ampicillin resistance.pJC792 and pJC831 were created by restriction digestion ofgel-purified 2.45-kb HindIII-EcoRI fragments by HindIII andClaI or MspI and subsequent ligation into pBR322 digestedwith Hindlll and ClaI or ClaI alone, respectively. StrainAB1157 was transformed to ampicillin resistance to obtainthe plasmids.

Derivatives of phage M13 were used in nucleotide se-quencing and are listed in Table 1 and Fig. 1A. PhagesJCM15295, JCM15298, and JCM15651 (Table 1) were con-structed by isolation of the indicated DNA fragment bypolyacrylamide gel electrophoresis and electroelution intodialysis bags (35). Such isolated fragments were added tovector replicative-form (RF) DNA treated by relevant re-striction nucleases. JCM15916 (Table 1) and other phages(Fig. 1A) were created by shotgun cloning. Source DNA andvector RF DNA were treated with cognate restriction endo-nucleases and ligase and then used to transform bacterialstrain JM103 or JM105 (37). Blunt-ended DNA fragmentswere ligated with Hindlll-cleaved M13 vector DNA. X revwas obtained as the lysogen N2262 from M. Gottesman. Theprophage was the Sam7 derivative of K rev ex ptsI describedby Gottesman et al. (21). A rev DNA, utilized in cloning intoM13 vectors, was prepared by a method modified from thatof Gillen et al. (20).DNA nucleotide sequence analysis of the 2.45-kb HindIll-

EcoRI fragments. Most sequencing of the 2.45-kb HindlIl-EcoRI fragment from pRAC1 was done by the chain termi-nation dideoxy method in accordance with the protocolsupplied in a kit purchased from New England BioLabs,Inc., Beverly, Mass. The method is based on that of Sangeret al. (39). Single-strand template from M13 phage wasprepared essentially in accordance with the protocol fromNew England BioLabs, a protocol modified from the methodof Schreier and Cortese (40). In most cases, the commer-cially available universal primer was used. To complete thesequence, however, we needed to obtain one unique primer,

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TABLE 1. Plasmids and M13 phages

Plasmid or Vector Insert(s)" Source Strain withphage" etrIsr() of insert plasmidc'

PlasmidspJC758 pBR322 2.45-kb" HindIlI-EcoRI fragment pRAC1 JC12592pJC792 pED825e 245-bp HindIII-ClaI fragment pJC758 JC13197pJC831 pBR322 Three MspI fragments of gel-purified 2.45-kb fragment pJC758 JC14708pJC843 pBR322 0.49-kbd HindlIl-EcoRI fragment pRAC3 JC14723pJC844 pBR322 1.14-kb" HindIII-EcoRI fragment pRAC4 JC14724pJC845 pBR322 1.54-kbd HindIII-EcoRI fragment pRAC5 JC14725pJC846 pBR322 0.42-kbd HindIll-EcoRI fragment pRAC7 JC04726pJC847 pBR322 0.55-kbd HindIII-EcoRI fragment pRAC10 JC14727pJC865 pBR322 7.9-kb ClaI-EcoRl fragmentf' SDB1041 JC15201pJC902 pBR322 7.9-kb Clal-EcoRl fragment' SDB10499 JC15608pJC929 pBR322 7.9-kb ClaI-EcoRI fragmentf' JC12020 JC15609pJC944 pBR322 7.9-kb ClaI-EcoRI fragmentf SDB10469 JC15614pSKM1 pBR322 8.1-kb' HindlIl fragment JC5412' JC15901

PhagesJCM15295 M13mp8 527-bp NruI-EcoRI fragment pRAC31 NSiJCM15298 M13mp8 537-bp BamHI-EcoRI fragment' pRAC36 NSJCM15651 M13mp9 537-bp BamHI-EcoRl fragment' pRAC36 NSJCM15916 M13mp8 1,711-bp EcoRI fragment pSKM1 NS

a Plasmids were detected by transforming AB1157 with a mixture ofDNA fragments treated with ligase, selecting for ampicillin-resistant transformants (9), andscreening crude DNA preparations (22) by restriction enzyme digestion and electrophoresis (35). Transformants were purified once before their plasmid DNA wasisolated for further use. Phages were detected by transfecting bacterial strain JM103 and detecting plaques on 5-bromo-4-chloro-3-indolyl-,-D-galactopyranosideas the indicator strain. Colorless plaques were picked. Crude preparations of RF DNA made from survivors in the plaque were screened by restriction nucleasedigestion followed by electrophoresis. Phages from appropriate plaques were single-plaque isolated before phage DNA was prepared for sequencing (37).

b All plasmid insert fragments plus that in JCM15916 were verified by restriction enzyme analysis of purified plasmid DNA. The remaining phage insertfragments were isolated by polyacrylamide gel electrophoresis before they were added to M13 DNA, which had been digested by cognate restriction nucleases.

All strains were derived from AB1157 (5), except JC15901, which was derived from JC5519, a recB21 recC22 derivative of AB1157 (5)."Sizes are estimated from the data of Willis et al. (47) and do not correspond exactly with the sizes determined in this work.epBR322 derivative (17).f This fragment consists of about 2.2 kb of Rac DNA and all of Tn5 (5.7 kb). pJC902 contains an additional uncharacterized 2.4-kb EcoRI fragment. pJC929

contains two additional uncharacterized EcoRI-ClaI fragments amounting to no more than 100 bp. The location of TnS in the Rac DNA is presented elsewhere(Chu and Clark, in preparation).

g See references 18 and 46.h Size estimated by Willis (Ph.D. dissertation).JC5412 is described by Clark (12) and Barbour et al. (7), although not by name. Kaiser and Murray (25) further characterized this spontaneous Uvr sbcA8

mutant of a recB21 strain.i NS, No strain.The same fragment isolated from other sources was used to make other phages used for nucleotide sequencing. Derivatives of M13mp8 were JCM15297

(source, pRAC2) and JCM15296 (source, X rev). Derivatives of M13mp9 were JCM15300 (source, pRAC2) and JCM15299 (source, X rev).

prJC8, complementary to positions 1556 to 1573. By arbi-trary convention, the first nucleotide of the HindIII recog-nition site in the HindIII-EcoRI fragment is designatedposition 1. The sequence of prJC8 is 5'-ATGCCAACCCTGATCCGG-3'; JCM15288 provided the template for thisprimer. Some sequencing was done by the chemical degra-dation method of Maxam and Gilbert (36). The insert frag-ment of pJC792 was sequenced by this method in bothdirections. First, pJC792 was digested with HindIII and PstI,resulting in two fragments, 1,023 and 1,477 base pairs (bp)long. The smaller fragment was gel isolated, 5' end labeledby a kinase exchange method modified from the methods ofBerkner and Folk (8) and Maniatis et al. (35), and digestedwith EcoRI, resulting in two fragments, 275 and 748 bp long.The smaller labeled fragment was gel isolated and se-quenced. Second, pJC792 was digested with ClaI, producinga linear 2,500-bp fragment. This fragment was gel isolated,5'-end labeled, and digested with PstI, resulting in twofragments, 1,727 and 773 bp long. The longer fragment wasgel isolated and sequenced. A 1,515-bp AvaI-EcoRI frag-ment from pJC758 that was 5' end labeled at the AvaI endwas sequenced by this method. A 550-bp EcoRI-HindIIIfragment from pJC831 that was 5' end labeled at the EcoRIend was subjected to this method to obtain the sequencefrom nucleotides 1214 to 1040 of the 2.45-kb HindIII-EcoRIfragment.

To determine whether the 2.45-kb HindIII-EcoRI frag-ment from pRAC1 contains a frameshift mutation, chaintermination dideoxy sequencing of single-stranded DNAfrom the phages listed in Table 1 was performed using twosynthetic primers. prJC9, whose sequence, 5'-GGCGCTGAACATCCGCAC-3', is complementary to positions 2176to 2193, was used to sequence single-stranded DNA fromour M13mp8 constructs. Similarly, prJC12, whose sequence,5'-CCGTAATAAATACCTGGC-3', is complementary topositions 2297 to 2280, was used to sequence single-strandedDNA from our M13mp9 constructs. In addition, usingprJC9, dideoxy sequencing of supercoiled plasmid DNAcontaining sbc::TnS mutations was performed as describedby Chen and Seeberg (10).To obtain the sequence of deletion junctions, DNAs of

pJC843, pJC844, pJC845, pJC846, and pJC847 were linear-ized with EcoRI, purified by passage through a NACS-52PREPAC minicolumn (Bethesda Research Laboratories,Gaithersburg, Md.) following the procedure suggested by themanufacturer, 5' end labeled, and digested with HindlIl,resulting in a labeled 4.33-kb pBR322 fragment and a labeledinsert fragment of a different size for each plasmid. Theinsert fragments were gel isolated and sequenced by thechemical degradation method.Computer analysis. Computer analysis of the DNA se-

quence was performed on a Sun Microsystems machine,

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A) INSERTS

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15280 15265 , ,

pJC792 15277 15273u 15279 ,1529 ~ 15275

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FIG. 1. Cloning and sequencing strategy for the 2.45-kb Hindlll-EcoRl fragment. The rectangle at the top is a partial restriction map ofthe HindIII-EcoRI fragment. The numbers above the map refer to the first base of the recognition sequence of various restriction enzymes.Those not labeled are TaqI sites. Only relevant XhoII, NruI, and MspI sites are labeled. Additional Xholl sites are found at coordinates 1934and 2253. Additional NruI sites are found at coordinates 1032 and 1944. Additional MspI sites are found at coordinates 539, 763, 910, 973,1348, 1570, 1714, 1821, 2029, 2161, 2327, 2368, 2451, and 2464. By using most Taql sites, the fragment can be divided into 11 regions as shown.(A) Inserts cloned into M13 vectors. Arrows indicate DNA fragments subcloned into M13 vectors; the tail of each arrow is proximal to theuniversal primer binding sequence. The vertical broken lines extending from restriction enzyme sites illustrate the boundaries of the clonedfragments. The numbers above the arrows, prefixed by JCM, are the names of the phage strains used for sequencing. The inserts can bedivided into two groups: (i) TaqI partial-digest fragments of the 2.45-kb HindIII-EcoRI fragment and (ii) fragments produced by digestion ofpJC758 with other restriction enzymes. TaqI fragments were ligated into the Accl site of M13mp19 vectors. The vector in JCM15280 isM13mp19. M13mp9 is the vector in JCM15259, JCM15265, and JCM15294. M13mp8 is the vector in JCM15291 and JCM15292. Inserts inM13mpl9 vectors were transformed into JM105 (48). All others were transformed into JM103 (37). (B) Sequence strategy. Arrows indicatethe extent sequenced 5' to 3'. A vertical broken line connects the beginning of each arrow to the restriction enzyme site where the DNAsequencing began. Chemical sequencing is indicated by an asterisk at the beginning of the arrow and by a pJC number above the arrow,denoting the plasmid utilized. Otherwise, dideoxy sequencing was employed. The numbers above these arrows, prefixed by JCM, indicatethe phages whose single-stranded DNA was employed for sequencing.

utilizing programs compiled by Hugo Martinez, Universityof California, San Francisco, and modified by others at theUniversity of California, Berkeley. Comparisons of DNAsequences were done by the FASTN program, utilizing theGenBank data base. Comparisons of amino acid sequenceswere done by the FASTP program, utilizing the NBRF-PIRprotein data base. Both of these comparison programs arebased on the work of Lipman and Pearson (31). Readingframes were analyzed by the program CODONUSE writtenin BASIC by C. Halling to implement the findings of Alff-Steinberger (2) and Staden (42). To predict protein second-ary structure, alpha-helix, beta-sheet, and beta-turn confor-mational parameter values for each amino acid of a primarystructure were calculated by the Fasman program, using themethod of Chou and Fasman (11) and the data set fromArgos et al. (4). These values were then interpreted byChou-Fasman rules (11). The rna_db program, based on thework of Zucker and Stiegler (49), was used to calculate theGibbs free energy (AG) of nucleic acid secondary structures.

Media, chemicals, and reagents. Luria broth (45) supple-mented with 50 ,ug of ampicillin per ml was used to select andmaintain plasmid-containing strains. 56/2 minimal medium(45) supplemented with 2% glucose was used to select and

maintain strain JM103 or JM105. Strains containing M13phage were propagated on Luria broth or YT or 2 x YTmedium (38).

Restriction endonucleases were purchased from New En-gland BioLabs and Bethesda Research Laboratories. Thereaction conditions used were those suggested by NewEngland BioLabs for high-, medium-, and low-salt buffers(15) except that bovine serum albumin was omitted. Primerswere prepared in the laboratory of J. Kirsch, University ofCalifornia, Berkeley, and were purified by thin-layer chro-matography (3).

RESULTS

Strategy for nucleotide sequencing. A 2.45-kb HindlIl-EcoRI fragment of pRAC1, hypothesized to encode theamino-terminal portion of ExoVIII, was cloned, withpBR322 as the vector. The resulting plasmid, pJC758, was

used as the source of DNA for subcloning. Purified HindIll-EcoRI fragment DNA was almost completely digested withTaqI nuclease. The RF of M13 phage strain mpl9 digestedwith AccI nuclease was used to clone TaqI fragmentsspanning 83% of the whole fragment (Fig. 1). The cloned

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1 AAGCTTGTGC CGAAAAAGAC CAGATGGCAC ATGAACTTTA ATTAATTGAC TATTCGAAAC TGAATTTATG CCAGAAATGG

81 CAGGTATTCG CTCAACCTTA ATTAAGGAGA AAAAC ATG ATT ACC AAT TAT GAA GCC ACT GTT GTA ACT ACC GATMet Ile Thr Asn Tyr Glu Ala Thr Val Val Thr Thr Asp

155 GAC ATT GTT CAC GAG GTG AAT CTG GAA &tA AAG CGC ATT GGC TAC GTA ATT AAA ACA GAA AAT AAA GAAAsp Ile Val His Glu Val Asn Leu Glu Gly Lys Arg Ile Gly Tyr Val Ile Lys Thr Glu Asn Lys Glu

188224 ACC CCA TTC ACT GTG GTT GAT ATC GAT GGT CCA TCA c5C AAC GTA AAA ACA CTT GAT GAA GGT GTC AAA

Thr Pro Phe Thr Val Val Asp Ile Asp Gly Pro Ser Gly Asn Val Lys Thr Leu Asp Glu Gly Val Lys

192293 AAA ATG TGC CTG GTG CAT ATC G%A AAG AAT CTG CCC GCA GAA AAA AAA GCC GAA TTT CTG GCA ACT CTA

Lys Met Cys Leu Val His Ile Gly Lys Asn Leu Pro Ala Glu Lys Lys Ala Glu Phe Leu Ala Thr Leu

362 ATT GCA ATG AAA TTA AAA GGT GAA ATC TGA AAGAA aTAGCCTGCG TATGG CAGIArTA GAACAGTGTGIle Ala Met Lys Leu Lys Gly Glu Ile ***

432 TATCCGGCAA GATCATTCAC TGAACAAAAC GAATTTTAAT CTGAGTTGAG GTTAAAAAAC A ATG AGC ACA AAA CCA CTCMet Ser Thr Lys Pro Leu

CTG TTA CGG AAALeu Leu Arg Lys

TCG ACC TGT GCCSer Thr Cys Ala

GTT GCC ACG AATVa 1 Ala Thr Asn

CGC TAT CAA CTCArg Tyr Gln Leu

TAT CAA GGC AATTyr Gln Gly Asn

GCGAla

ACTThr

TTTPhe

AGCSer

ACCThr

AAA AAALys Lys

CTG GACLeu Asp

CCT GTCPro Va 1

AAA GACLys Asp

AAC GTCAsn Val

TCA TCC GGTSer Ser Gly

TAC CTG ATCTyr Leu Ile

GTT AAT GACVal Asn Asp

TCC ATG ACASer Met Thr

AAC GGC GAAAsn Gly Glu

GAA CCTGl u Pro

GTT AAGVa 1 Lys

CTG CCCLeu Pro

TGG GAATrp Gl u

GAC ATGAsp Met

GAC GTC GTCAsp Va1Val

TCA GGT AAASer Gly Lys

GCT GAA GGTAla Glu Gly

CTA AAA CCGLeu Lys Pro

ACT GAG ATTThr Glu Ile

CTG TGG GCA AGC AAC GAT TTTLeu Trp Ala Ser Asn Asp Phe

AAA CTG AGC AGC TAT TTT AAALys Leu Ser Ser Tyr Phe Lys

GAG ATC GAT TTT ACC TGG AGTGlu Ile Asp Phe Thr Trp Ser

GGA GCA GCA CCA GAC AAC GCTGly Ala Ala Pro Asp Asn Ala

GAG GAG AAT ATG CTA CTC CCAGlu Glu Asn Met Leu Leu Pro

856 ATT TCT GGC CAG GAAIle Ser Gly Gln Glu

925 TCA CGCSer Arg

994 GTT TCCVal Ser

1063 GAC AAAAsp Lys

1132 AAC GCTAsn Ala

1201 ACT CGCThr Arg

GACAsp

CACHis

GTTVal

GACAsp

ACGThr

1270 GAT CTG ACGAsp Leu Thr

1339 AAC CTT CATAsn Leu His

1408 TTC CGC GACPhe Arg Asp

GGA CTCGly Leu

AAA ACCLys Thr

189CTG&CCCLeu Pro

CAG GCAGln Ala

AGC CTGSer Leu

TTC CCT AAT CCTPhe Pro Asn

TAC ACC GATTyr Thr Asp

GCT TCC GGTAla Ser Gly

Pro

CGAArg

GCTAla

190TCA CTG GCG!CGCSer Leu Ala Arg

CCG GCA CAC GCTPro Ala His Ala

AAA TTC ATC ACCLys Phe Ile Thr

ATTIle

TTALeu

CTCLeu

GGTGly

GGAGly

AATAsn

CGT TGG CTT GCT CAAArg Trp Leu Ala Gln

CAC ATT GCT CGG GCTHis Ile Ala Arg Ala

GAC CCG CTG GAA ATTAsp Pro Leu Glu Ile

AAT TCA AAC CTG GGAAsn Ser Asn Leu Gly

CTG CTG ACA AAA GAGLeu Leu Thr Lys Glu

GCT GGC GGC GGA AACAla Gly Gly Gly Asn

GAC GTA GCC ACT GGC GTAAsp Val Ala Thr Gly Val

AAA CGC ATT GAG GAA ATTLys Arg Ile Glu Glu Ile

ATG CCT GGC GGG CTG GATMet Pro Gly Gly Leu Asp

CAC GGC AGC GAAHis Gly Ser Glu

GAA GAA CTA CCGGiu Glu Leu Pro

CGC GAA CTC CACArg Glu Leu His

CTG ATA ACT GCTLeu Ile Thr Ala

TGG ATG AAG GGTTrp Met Lys Gly

CTC ACC GAT CGCLeu Thr Asp Arg

CTG GCC CGT TCALeu Ala Arg Ser

ATC GCT GAA AATIle Ala Glu Asn

TAT TCC CGC GCCTyr Ser Arg Ala

AAA CCG GTA ACG CAC GTTLys Pro Val Thr His Val

GCT GTT ACT GCC CTG GCTAla Val Thr Ala Leu Ala

AAA CTG GTT CGT GAC ACTLys Leu Val Arg Asp Thr

TTT TTC GAA GCA TAC CTGPhe Phe Glu Ala Tyr Leu

AAT CGT GTT TCA CAC ATCAsn Arg Va 1 Ser His Ile

GGC GAA GGT TTC GTA CACGly Glu Gly Phe Val His

ATG GAT CTG GAC ATC TATMet Asp Leu Asp Ile Tyr

AAA CCG CCC TTT TCT GTTLys Pro Pro Phe Ser Val

ATC GTG GTT GCG TCC GTAIle Val Val Ala Ser Val

TaqI fragments divide the 2.45-kb HindIII-EcoRI fragmentinto 11 regions with 10 junctions between regions. Theseclones were used to obtain single-stranded DNA for dideoxysequencing of both strands of regions 2 through 10, except

region 5. The amount ofDNA sequence obtained by dideoxysequencing of these and other clones and by chemicalsequencing is summarized in Fig. 1B. To complete thesequence of the junctions and of the regions of DNA that

511 TTCPhe

580 GAAGl u

649 GCTAla

718 GAAGl u

787 CACHis

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1477 AAA GAA GCA CCA ATT GGG ATC GAG GTC ATC CCC GCG CAC GTC ACT GAA TAT CTG AAC AAA GTA CTG ACTLys Glu Ala Pro Ile Gly Ile Glu Va1 Ile Pro Ala His Va1 Thr Glu Tyr Leu Asn Lys Val Leu Thr

1546 GAA ACC GAT CAT GCC AAC CCT GAT CCG GAA ATC GTG GAT ATT GGC TGC GGT CGC TCC TCT GCC CCG ATGGlu Thr Asp His Ala Asn Pro Asp Pro Glu Ile Val Asp Ile Gly Cys Gly Arg Ser Ser Ala Pro Met

1615 CCG CAG CGA GTA ACA GAA GAA GGA AAA CAG GAT GAT GAA GAA AAA CCG CAA CCA TCT GGA ACA ACG GCAPro Gln Arg Val Thr Glu Glu Gly Lys Gln Asp Asp Glu Glu Lys Pro Gln Pro Ser Gly Thr Thr Ala

1684 GTT GAA CAG GGA GAG GCT GAA ACA ATG GAA CCG GAC GCA ACT GAA CAT CAT CAG GAC ACG CAG CCG CTGVal Glu Gln Gly Glu Ala Glu Thr Met Glu Pro Asp Ala Thr Glu His His Gln Asp Thr Gln Pro Leu

1753 GAT GCT CAG TCA CAG GTA AAT TCT GTT GAT GCG AAA TAT CAG GAA CTG CGG GCA GAA CTC CAT GAA GCCAsp Ala Gln Ser Gln Val Asn Ser Val Asp Ala Lys Tyr Gln Glu Leu Arg Ala Glu Leu His Glu Ala

1822 CGG AAA AAC ATT CCA TCA AAA AAT CCT GTC GAT GAC GAT AAA TTG CTT GCT GCA TCA CGT GGT GAA TTTArg Lys Asn Ile Pro Ser Lys Asn Pro Val Asp Asp Asp Lys Leu Leu Ala Ala Ser Arg Gly Glu Phe

1891 GTT GAC GGA ATT AGC GAC CCG AAC GAT CCG AAA TGG GTA AAG GGG ATC CAG ACT CGC GAT TGT GTG TACVal Asp Gly Ile Ser Asp Pro Asn Asp Pro Lys Trp Val Lys Gly Ile Gln Thr Arg Asp Cys Val Tyr

1960 CAG AAC CAG CCA GAA ACG GAA AAA ACC AGC CCA GAT ATG AAT CAA CCT GAG CCA GTA GTG CAA CAG GAAGln Asn Gln Pro Glu Thr Glu Lys Thr Ser Pro Asp Met Asn Gln Pro Glu Pro Val Val Gln Gln Glu

2029 CCG GAA ATA GCC TGC AAT GCC TGC GGC CAG ACT GGC GGG GAT AAC TGC CCT GAC TGT GGT GCG GTG ATGPro Glu Ile Ala Cys Asn Ala Cys Gly Gln Thr Gly Gly Asp Asn Cys Pro Asp Cys Gly Ala Val Met

2098 GGC GAC GCA ACA TAC CAG GAA ACA TTC GAT GAA GAG AGT CAG GTT GAA GCT AAG GAA AAT GAT CCG GAGGly Asp Ala Thr Tyr Gln Glu Thr Phe Asp Glu Glu Ser Gln Val Glu Ala Lys Glu Asn Asp Pro Glu

2167 GAA ATG GAA GGC GCT GAA CAT CCG CAC AAT GAG AAT GCT GGC AGC GAT CCG CAT CGC GAT TGC AGT GATGlu Met Glu Gly Ala Glu His Pro His Asn Glu Asn Ala Gly Ser Asp Pro His Arg Asp Cys Ser Asp

9 19 1912236 GAA A;TGliu Thr Gly

118 189GAA GTC GCA AT TCCGlu Val Ala Asp Pro

GTA ATC GTA GAA GACVal Ile Val Glu Asp

1$2190ATA G'AGI:CAIle Giu Pro

GGT ATT TAT TAC GGA ATT TCGGly Ile Tyr Tyr Gly Ile Ser

2305 AAT GAG AAT TAC CAC GCG GGT CCC GGT ATC AGT AAG TCT CAG CTC GAT GAC ATT GCT GAT ACT CCG GCAAsn Glu Asn Tyr His Ala Gly Pro Gly Ile Ser Lys Ser Gln Leu Asp Asp Ile Ala Asp Thr Pro Ala

2374 CTA TAT TTG TGG CGT AAA AAT GCC CCC GTG GAC ACC ACA AAG ACA AAA ACG CTC GAT TTA GGA ACT GCTLeu Tyr Leu Trp Arg Lys Asn Ala Pro Val Asp Thr Thr Lys Thr Lys Thr Leu Asp Leu Gly Thr Ala

2443 TTC CAC TGC CGG GTA CTT GAA CCG GAA GAA TTCPhe His Cys Arg Val Leu Glu Pro Glu Glu Phe

FIG. 2. DNA sequence of the wild-type 2.45-kb HindII-EcoRI RacC fragment. One strand of sequence is presented 5' to 3', in which thefirst base of the Hindlll site is coordinate 1 and the first base of the EcoRl site is therefore coordinate 2470. Nucleotide coordinates areprovided on the left of the sequence. The racC gene, with the deduced amino acid sequence shown, begins at nucleotide 116 and terminatesat an opal codon ending at nucleotide 391. The recE gene, with the deduced amino acid sequence shown, begins translation at nucleotide 493and continues through the end of the fragment. A dyad symmetry spanning positions 397 to 421 is underlined. The locations of A(recE) andA&(racC-recE) deletion endpoints are marked with closed inverted triangles, labeled with the allele numbers, between the nucleotides at thedeletion junctions. The frameshift deletion, recE939, is marked by an open triangle, labeled with the allele number, positioned over thenucleotide that is removed by this mutation.

remained unsequenced after dideoxy sequencing of theseTaqI fragment clones, a combination of chemical and dide-oxy sequencing was employed.As described below, we found that pRAC1 contained a

mutant allele of recE. To determine and confirm the wild-type sequence, we used nine other sources of recE DNA:pRAC2, X rev, two sbcA mutants, an sbc-23 point mutant,and four sbc::TnS mutants (Table 1). In each case only asmall region near the EcoRI site was sequenced. WithM13mp8 and M13mp9 as vectors, this region from pRAC2, Xrev, and the sbcA6 mutant was subcloned as a 537-bpBamHI-EcoRI fragment (Table 1). It was similarly sub-cloned as a 527-bp NruI-EcoRI fragment from the sbc-23mutant and as a 1,711-bp EcoRI fragment from the sbcA8mutant (Table 1). For sequencing the region from the four

sbc::TnS mutants, we used plasmids pJC865, pJC902,pJC929, and pJC944 (Table 1).

Nucleotide sequence. The nucleotide sequence of the wild-type HindIII-EcoRI fragment consists of 2,475 bp, whichagrees well with the estimate of 2.45 kb based on gelelectrophoresis (14, 47). Two open reading frames (ORFs)can be translated in the same direction from HindIIl toEcoRI (Fig. 2). One ORF extends from nucleotides 50 to 388.Three ATG codons exist within the first 70 nucleotides ofthis ORF. The third, at nucleotide 116, seems the most likelyinitiating codon because of the nature of its potential ribo-some-binding site. Four bases upstream of this ATG thesequence 5'-TAAGGAGAA-3' begins. The first seven ofnine nucleotides are complementary to the 3' end of the 16SrRNA (41). We have called this ORF from the ATG at 116 to

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TABLE 2. Endpoints of deletion mutations which suppress recE939"

size ofallele deletionnumber plasmid (in bp) upstream ORF of recE downstream ORF of recE

126 127 ' 28 129 1 301q22 -r.AA c(T(. : CCC: ATT CCT-

993

2060

1994

1964

5 6 °7 8 9£,CA GAT: CCC GTA ATC-

264 26°5 2:66 267 268 13 14; 1;5 16 17-CTG GCG Ck,C GAC GTA- -ATA GAG CCA GGT ATT-

racC downstream ORF of recE

21 22 2 3-i 24 25 1 '2 3 4-CTG GAA GG'A AAG CGC- -TGA AAT ;G G;C GAA GTC-

47 4i84i;9 50 51 4 5; 7 8-CCA T!CA GIGC AAC GTA- -GTC G:CA G,AT CCC GTA-

65 66 6 7 68 69 12 13 riFf*4 15 16-CAT ATC_GIGA AAG AAT- -GAC ATA YAGCCA GGT-

" Identical sequences at the endpoints are enclosed by broken boxes. Such identities create ambiguity as to the exact endpoints. We arbitrarily chose endpointsby using the criterion of having a G on the left-hand side of the novel junction. The open rectangles indicate the deleted bases; the closed rectangles indicate thebases that form the novel joint. Another equally good criterion would have been to break the phosphodiester bonds between adjacent GC (or CG) base pairs toform the novel joint. Using that criterion, we would have shifted the junctions of deletion 189 and deletion 190 one nucleotide to the right. The full names of thesedeletion alleles are A(recE)189, A(recE)190, A(racC-recE)191, A(racC-recE)188, and A(racC-recE)192. The estimated sizes of the deletion alleles, from Willis etal. (47), are as follows: 189, 1.31 kb; 190, 0.91 kb; 191, 2.03 kb; 188, 1.96 kb; 192, 1.90 kb. Codons in the sequence are numbered.

the TGA ending at 391 the racC gene (14). Support for thehypothesis that this gene is translated comes from threesources. First, analysis of codon usage in this ORF by theprogram CODONUSE indicated that it is translated. Sec-ond, deletion analysis showed that an 11-kDa protein ismade from this region (14). Our sequence predicts a proteinof about 10 kDa, a fairly close agreement. Third, theamino-terminal sequence of a racC-recE fusion protein cor-

responds with the first 16 amino acids predicted for racCfrom the DNA sequence (34).A strong dyad symmetry is present between racC and the

second ORF. A perfect 10-bp stem with a 5-bp loop found atpositions 397 to 421 has a predicted AG of -22.20 kcal (ca.-92.86 kJ). No other strong dyad symmetries and no notabledirect repeats are present in the whole HindIII-EcoRI frag-ment.The second ORF extends from nucleotide 487 to the

EcoRI cleavage site (Fig. 2). The program CODONUSEindicated that this ORF is translated. A possible translationalstart exists at position 493. Nine nucleotides upstream of theATG at this position lies the sequence 5'-GAGGT-3', a

sequence which is perfectly complementary to the 3' end ofthe 16S rRNA. Although, the ribosome-binding site is usu-

ally 4 to 7 nucleotides upstream of the translational start site,it has occasionally been found up to 13 nucleotides upstream(43). The amino-terminal sequence of the first 12 amino acidsof ExoVIII agrees with the sequence of amino acid codonsdownstream of the ATG at 493, indicating that this ORF isrecE and that the initiating methionine is removed from thefinal product (34). In analysis with the FASTP program, theamino acid sequence derived from the first 661 codons ofrecE showed no significant similarity with any protein se-

quences in the NBRF-PIR 15.0 (December 1987 data base),including lambda exonuclease. This is consistent with pre-

vious findings that ExoVIII has activities identical to thoseof lambda exonuclease but does not cross-react with anti-body raised against lambda exonuclease (19, 29).

A recE frameshift mutation and its deletion suppressors. Weinitially sequenced DNA from plasmid pRAC1, which doesnot contain an active recE gene (47). In that DNA we foundone fewer base pair than in the sequence shown in Fig. 2.The CG base pair at position 2240 was missing, thus termi-nating the putative recE reading frame seven codons down-stream. We named the mutation in pRAC1, recE939.

Willis et al. (47) described five deletion mutations ofpRAC1 which led to the production of ExoVIII activity.These deletions suppress the recE939 mutation of pRAC1.We located their endpoints by the chemical method ofsequencing. The locations are indicated in Fig. 2, and theircharacteristics are summarized in Table 2. The sizes of thedeletions determined by sequencing are in close agreement(within 100 bp) with those determined previously by gelelectrophoresis (47). All five deletions remove recE939 andfuse the downstream portion of recE to upstream readingframes which contain translation initiating signals (Table 2).Two deletions start and end within recE and have beennamed A(recE)189 and l(recE)J90. (Operating under thehypothesis that the deletions removed a sequence which was

altered by sbcA point mutations, Clark et al. [14] had namedthese deletions A(sbcA)188, A(sbcA)189, etc. Since thishypothesis was disproved by this work, these names are nolonger operative. The new names retain the allele number;only the parenthetical expression has been changed.) Threedeletions start within racC and end within recE. In confor-mity with standard practice we named these mutationsA(racC-recE)188, A(racC-recE)J91, and A(racC-recE)I92.The ExoVIII activity was purified from a strain carryingA(racC-recE)188, and the amino-terminal amino acid se-

quence was determined (34). The first 16 amino acids of thisprotein correspond perfectly with the expected amino acidsequence of the racC protein (34). Thus, the RecE activityproduced by pRAC1 deletion derivatives can be explainedby a removal of recE939 and the subsequent translation ofthat part of recE encoding the carboxy terminus.

pRAC4

pRAC5

189

190

191

188

192

pRAC7

pRAC3

pRACIO

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SUPPRESSION OF recE MUTATION 2107

DISCUSSION

Using a lambda phage strain as the vector, Kaiser andMurray (26) had cloned recE, the gene encoding ExoVIII, aspart of a 7.6-kb HindIII fragment derived from an sbcA+strain of E. coli. The hybrid phage was selected for expres-sion of recE by its ability to form plaques on polA and ligmutant hosts. Using pBR322 as the vector, Willis et al. (47)cloned a similar fragment from sbcA+ strain KL16, usingnonselective colony hybridization to detect hybrid plasmids.One of the two plasmids obtained, pRAC2, also containedextraneous fragments and was set aside. The other, pRAC1,did not express recE, as judged by the absence of ExoVIIIactivity and a protein of the size of ExoVIII (14, 47). Weshowed in this study by nucleotide sequence analysis thatthis region from pRAC1 contains a single base pair deletionin codon 583 of recE, which we call recE939. This deletiontruncates translation of the recE reading frame seven codonsdownstream. Deletion from pRAC1 of a region betweenEcoRI and HindlIl restriction endonuclease cleavage sitesand replacement with the same region from the chromo-somes of sbcA6 and sbc-23 mutants led to two plasmids,pRAC26 and pRAC31, which produced functional recE. Thiswas interpreted to reveal the location of sbcA mutations (14,47). We showed that this fragment replacement restored thewild-type recE sequence and conclude that it does not revealthe location of sbcA.recE939 may have been present as a preexisting mutation

in the population of KL16 from which Willis et al. (47)obtained their DNA. Alternatively, the mutation may haveoccurred subsequent to cloning. Isolation of a recE+ deriv-ative of pBR322 in the original screening (i.e., pRAC2) andconversion of pRAC1 to a recE+ derivative by EcoRI-Hindlll fragment replacement (pRAC26 and pRAC31) makeit seem unlikely that functional recE is of selective disadvan-tage when present on pBR322. Thus, we have no basis forpreferring preexistence or subsequent occurrence as alter-native hypotheses for the origin of recE939.

Using the FASTN program we found that the nucleotidesequence of the HindIII-EcoRI fragment shows no obvioussimiiarity to sequences in GenBank 54.0 (December 1987),including that of lambda phage. This is consistent with theabsence of heteroduplex formation between this region ofRac and the region of lambda it replaces to make rev (21).In the recE+ sequence there are two isodirectional ORFsseparated by a short region containing dyad symmetry. Wehypothesize that this dyad sequence serves a regulationalfunction, perhaps as a transcriptional terminator or a repres-sor-binding site. If it is a terminator, it must be less than100% effective, because Willis et al. (47) showed that recE+was expressed from one or more promoters in the pBR322cloning vector; hence, some transcription would have topass the dyad symmetry.

According to the nucleotide sequence, the truncatedrecE939 protein should have a mass of about 65 kDa.Analysis by sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis of the recE939 protein shows a mass ofabout 86 kDa, however (14, 34). Reduced mobility in SDS-polyacrylamide gels is often due to resistance of the proteinto SDS binding, which reduces the net charge on the protein,resulting in slower gel electrophoresis and an apparenthigher molecular mass. A protein structure consisting pre-dominantly of a beta-sheet may be resistant to SDS binding,as is the P22 gene 9-encoded tail spike protein (27). Chou-Fasman analysis of the predicted amino acid sequence of therecE939 peptide shows a series of five alpha-helix and five

beta-sheet sequences, alternating for the most part, fromamino acids 257 to 355. This region is strikingly the densestregion of beta-sheets predicted by Chou-Fasman analysis ofthe sequence. Thus, on the basis of a predominantly beta-sheet structure, the region from amino acids 257 to 355 mightbe expected to be SDS binding resistant and hence causereduced mobility in SDS-polyacrylamide gels.We believe recE+ protein also migrates anomalously in

SDS-polyacrylamide gels. Preliminary nucleotide sequencedata show that the C terminus of recE would be at position3895 if the sequence in Fig. 2 were extended (C. Chu, V.Sharma, S. Abremowitz, L. Satin, and A. J. Clark, unpub-lished results). The mass of the recE+ protein estimatedfrom the preliminary sequence is 120 kDa. Analysis bySDS-PAGE yields an estimate of 140 kDa for ExoVIII (e.g.,references 14, 28, and 34). We believe the difference of 20kDa is contributed by the amino-terminal region fromcodons 1 to 583, shared in common with the recE939 protein.Support for this hypothesis comes from analysis of the sizesof proteins made by the pRAC1 deletions. Luisi-DeLuca etal. (34) measured the sizes of these proteins by Western blot(immunoblot) analysis, using antibodies to ExoVIII. Thesizes they observed were consistent with deletion or disrup-tion of the domain responsible for reduced mobility inSDS-polyacrylamide gels. The smallest deletion removedcodons 266 to 590 and would have disrupted the hypothe-sized SDS-resistant domain (codons 257 to 355).Our analysis showed that about 70% of the sequenced

region of recE could be deleted without inactivating Ex-oVIII. Two mutations (188 and 191; Table 2) which lead tothe largest amounts of ExoVIII activity as determined byWillis et al. (47) remove more than 580 codons of recE andfuse in frame the C-terminal portion of recE to the N-terminal portion of racC. Amino acid sequence data support-ing this conclusion are presented by Luisi-DeLuca et al. (34)for the A(racC-recE)188 protein. Expendability of the amino-terminal region leads us to suggest that it encodes at leastone protein domain. Consequently, the portion of recE fromthe initial ATG (nucleotides 493 to 495) to recE939 (nucleo-tide 2240), we designated domain 1. Accordingly, domain 2of recE would encode the nuclease domain of the recE+protein and reside in the carboxy-terminal half of the pro-tein. Luisi-DeLuca et al. (34) and V. Sharma and A. J. Clark(unpublished results) have evidence consistent with thehypothesis that there are actually three domains in recE.Potential boundaries in the carboxy-terminal half of ExoVIIIbetween domains 2 and 3 are discussed by Luisi-DeLuca etal. (34).The left-hand endpoints of five deletions which remove

recE939 span 1,100 nucleotides, whereas the right-handendpoints span just 26 nucleotides (Fig. 2 and Table 2).Clustering of the right-hand endpoints could reflect either ashort distance between recE939 and a crucial coding regionof the nuclease domain or a special mechanism of deletionformation. We found no special sequences defining the endsof the deletions, however. In addition, the short regions ofnucleotide identity at the junction points (Table 2) make ourdeletions similar to the rare class of spontaneous deletionsdescribed by Albertini et al. (1). Thus, we do not favor thespecial mechanism hypothesis. On the other hand, becausemuch more nuclease activity is produced by deletion alleles191 and 188 than by the other three deletion alleles (34, 47)and because this correlates with the removal of less recEdownstream of recE939 by alleles 191 and 188 (Table 2), weprefer the hypothesis that the nuclease domain begins withina few codons of recE939.

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2108 CHU ET AL.

ACKNOWLEDGMENTS

We especially thank the following: Conrad Halling, who gra-

ciously adapted his CODONUSE program for the IBM PC AT andIBM Proprinter and constructed pJC854-7 during his laboratoryrotation; Michael Blanar, who tapght C.C.C. chemical sequencingand computer techniques; Warren Gish and Mike Cherry, who were

helpful computer wizards; Hans "Hulk" 'Cheng, who initiatedsequencing of pJC854-7; Cindy Luisi-DeLuca and Richard Kolod-ner, who provided thoughtful discussion and shared unpublisheddata; members of the laboratory of A.J.C., especially Ann Templin,who were all a tremendous help in the laboratory; and God, whomade all things possible.

C.C.C. was supported through the Department of Genetics byNational Institutes of Health Predoctoral Institutional TrainingGrant GM07127. This work was supported in part by Public HealthService grant A105371 from the National Institutes of Health.

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