Vol. 173, No. 3JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1208-12140021-9193/91/031208-07$02.00/0Copyright C) 1991, American Society for Microbiology

Transcription and Initiation of ColEl DNA Replication in

Escherichia coli K-12NAOKI INOUEt* AND HISAO UCHIDAt

Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan

Received 7 December 1989/Accepted 1 December 1990

By Sl nuclease protection mapping, we characterized RNA transcripts and nascent ColEl DNA synthesizedin wild-type Escherichia coli cells after infection with X-mini-ColEl hybrid bacteriophages. Transcription ofthe RNA II region of ColEl DNA in vivo starts mostly from the RNA II promoter, which was identified by invitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColEl DNA was found tostart at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending atthe ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of thatof the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the numberof transcripts ending at the on site was much reduced and that of transcripts reading through the on site wasdefinitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of theRNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system,but most of the cleaved RNA is unable to prime initiation of ColEl DNA synthesis efficiently.

We have previously shown that the replication-defectivecer-6 mutant, as well as wild-type ColEl DNA, replicates inEscherichia coli K-12 polA' cells lacking RNase H (20, 22).Furthermore, Kogoma (16) showed that the ColEl plasmid ismaintained in an rnh polAl strain. On the other hand, resultsof in vitro studies strongly suggested that the role of RNaseH is to cleave an RNA transcript (RNA II) and the cleavedproduct is used as the primer to initiate DNA synthesis at thecleavage site (11, 12). In contrast to the large amount ofinformation available on in vitro transcription from ColElDNA, we know little about in vivo transcription of the DNA.Therefore, in this study, we characterized RNA transcriptsand nascent DNA synthesis after infection of E. coli cellswith A bacteriophages containing a mini-ColEl genome (24)to see whether the picture obtained from the in vitro systemusing purified enzymes explains the in vivo situation.

Recently, Dasgupta et al. (3) presented a multiple-mecha-nism model which assumes different mechanisms of initia-tion of replication to explain the behavior of various mutantbacteria. This model is based mainly on evidence obtainedby analyses of nascent DNA synthesized in crude extractsdepleted ofRNase H or DNA polymerase I. They postulatedthat in wild-type bacteria, primer formation follows thepreviously proposed model (type I mechanism); that inbacteria lacking both RNase H and DNA polymerase I,hybridization between template DNA and readthrough tran-scripts leads to displacement of a single-stranded region ofthe nontranscribed strand on which lagging-strand synthesistakes place (type II mechanism); and that in bacteria lackingonly RNase H, transcripts extending beyond the ori sitespontaneously terminate at multiple sites and are used asprimers for leading-strand synthesis at these sites withoutfurther cleavage by RNase H (type III mechanism). Tomi-zawa and Masukata (26) observed that spontaneous termi-

* Corresponding author.t Present address: Department of Virology and Rickettsiology,

National Institute of Health, Shinagawa-ku, Tokyo 141, Japan.t Present address: Department of Biosciences, Teikyo Univer-

sity, Utsunomiya 320, Japan.

nation of transcription in vitro at the ori site or within 50nucleotides (nt) downstream from the ori site was rare, butthey postulated different efficiencies of utilization by DNApolymerase I among hybridized transcripts, so that thoseextending for a short distance (less than 50 nt) beyond thenormal replication origin are used efficiently as primers,while those extending further downstream are not usedefficiently unless cleaved by RNase H (3). What we wouldlike to know is whether transcription terminates in vivo at ornear the ori site and how frequently such termination occursin bacteria lacking RNase H.

In this study, we detected and identified transcripts andnascent DNA synthesized by the ori region of ColEl in E.coli. For mapping, we modified the procedure of Berk andSharp (1) by using homogeneously 32P-labeled M13 tem-plates containing various fragments of the ColEl ori region.The procedure allowed us to identify the polarity and extentof newly synthesized nucleic acids.

MATERIALS AND METHODS

Bacterial strains and phages. The E. coli K-12 strains usedand their relevant genetic characteristics are as follows: 594(supo strA) (2); JM101 (17); AB301-RNase-19 (rna) (5);AB301-105 (rna rnc) (15); SN50 (her') and SN51 [herA(=rnh)39(Ts)] (20); SN296 [herA136(Am) supo] (21); ON112(rnh+); ON121 [rnh-59(Ts)] and ON152 (rnh-91) (23).MIC1001 (rnh+) and MIC1020 (rnh-339::cat) (10) werekindly given by R. J. Crouch. Xind (13) was used to constructlysogens. XSN4 is a X-mini-ColEl hybrid phage, andXSN4cer-6, XSN4cer-10, and XSN4cer-17 are ColEl replica-tion-defective mutants (20). M13mp2 (7) and M13mpll (18)were used to construct probe DNAs.Media and buffers. CA broth contained 1% Casamino

Acids (Difco), 0.25% NaCl, and 1 ,ug of thiamine chloride perml, pH 7.0. TY/Mal broth contained 1% Tryptone (Difco),0.1% yeast extract (Difco), 0.8% NaCl, and 0.2% maltose.SSC buffer (lx) was 0.15 M NaCl plus 15 mM sodiumcitrate. TM buffer was 10 mM Tris hydrochloride (pH 7.4)containing 10 mM MgSO4.

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INITIATION OF ColEl DNA REPLICATION IN E. COLI 1209

Preparation of 32P-labeled single-stranded DNA probes.32P-labeled M13 phage was prepared by infection ofJM101in low-phosphate CA broth which contained 5,ug of Pi per mland 50 to 250,uCiof 32Pi per ml. M13 phage particles wereconcentrated by precipitation with polyethylene glycol 6000and purified by CsCl equilibrium centrifugation. Single-stranded DNAs were extracted from M13 phage particleswith phenol.

Preparation of nucleic acids from cells infected with X-mini-ColEl phages. Cells were grown to 3 x 108/ml in 100 ml ofTY/Mal broth, harvested by centrifugation, and suspendedin 1.5 ml of TM buffer. After adsorption of X-mini-ColE1phage at a multiplicity of infection of5 for 15 min, the cellswere added to 20 ml of prewarmed CA broth and incubatedwith aeration. At a designated time, the cells were mixedwith 20 ml of crushed frozen medium which contained 10mM Tris hydrochloride (pH 7.4), 1 mM EDTA, 20 mMsodium azide, 40,ug of rifampin, and 250,ug of chloramphen-icol powder per ml and collected by centrifugation. Theprecipitated cells were suspended rapidly in lx SSC buffercontaining 2% sodium dodecyl sulfate and boiled for 2 min.Nucleic acids were then extracted three times with SSC-saturated phenol at 60°C, twice with SSC-saturated phenol-chloroform (1:1, vol/vol), and once with ethyl ether. Sampleswere digested with HindIll to reduce their viscosity. Thereis no HindlIl recognition site in the ori region of the ColElreplicon. When necessary, nucleic acids were then treatedwith either RNase A (Sigma) which had been heated at 90°Cfor 2 min or RNase-free DNase I (Worthington). Overallefficiency of extraction was higher than 95% for RNA andabout 50% for DNA.

Si mapping analysis. S1 mapping analysis was performedby the method of Berk and Sharp (1) with some modifica-tions. Nucleic acids (100 to 300 ,ug), equivalent to about 3 x109 cells, and 32P-labeled single-stranded DNA (2 x 105 to 5x 105 cpm) were mixed into 50 ,ul of buffer containing 80%formamide, 40 mM piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES; pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Themolar amount of M13 probes was a 20-fold excess over thatof the infecting X DNA. After denaturation by heating at83°C for 10 min, the mixture was incubated at 39°C for atleast 12 h. The reaction was terminated by dilution with 400,ul of buffer containing 50 mM NaCl, 30 mM sodium acetate(pH 4.6), and 1 mM ZnSO4. S1 nuclease (100 to 300 U; BRL)was added, and the sample was incubated at 45°C for 30 min.It was then precipitated with ethanol. The precipitate wassuspended with 100 RI1 of 0.1 M NaOH-60 mM EDTA andincubated at 68°C for 30 min to degrade the RNA. Afterneutralization, it was precipitated with ethanol and sus-pended in 5 ,ul of 80% formamide-0.1 M NaOH-0.05%bromophenol blue. After incubation at 93°C for 1 min, it waschilled quickly and loaded on a 5% (or 6%) polyacrylamidegel containing 8 M urea for electrophoresis. Radioactivebands were detected by autoradiography. The chain lengthof each band was estimated by comparison with appropriatesize standards, and the size of the nucleic acid was desig-nated in terms of the number of nucleotides. The relativestandard deviation (cr) of chain length determination wasestimated as 0.35% between 200 and 600 nt. Amounts ofnucleic acids represented by each band were estimated bymicrodensitometry of autoradiographs with ATTO, ACD-25X, and expressed in terms of molar quantity relative to theinfecting A phage DNA present in the sample. The lowerdetection limit was less than 0.05 copy per infecting A DNAin most of the experiments.

EcoRI(+4U)

A"c(+VI)

Avail(-62)

AeII-Ih)

Mboll(+l) Mboll(-62)

EcoRl(+I) ERI(-4)oo+400.1 -eoo-100or

2A/2B

3A/3B

4A/4B5A/5B

pAO3

FIG. 1. Probes for Si nuclease protection mapping. pAO3 orpAO7 DNA was digested with the restriction enzymes indicated,repaired with the large fragment of DNA polymerase I, and ligatedwith an 8-nt EcoRI linker. Each fragment was then cloned into theEcoRI site of M13mp2 or M13mpll. A-type probes detected tran-scripts or DNA strands having the same polarity as the leadingstrand of pAO3 replication, and those detecting the reverse polaritywere referred to as B types. The probes shown were designated 2Athrough SA and 2B through SB. The position of a particularnucleotide on pAO3 was designated by the number of nucleotidesfrom the ori site, with a plus or minus sign, depending on whetherthe position was downstream or upstream from the ori site. Num-bers after restriction sites indicate positions of fragment ends.

Nucleotide sequence accession number. Miniplasmid pAO3has been assigned GenBank/EMBL accession no. J01566.

RESULTSMapping of RNA transcription and DNA replication of the

wild-type ColEl replicon. Nucleic acids synthesized afterinfection of E. coli cells with various A-mini-ColE1 phageswere extracted and analyzed. A A-mini-ColE1 phage calledASN4 was described previously (21). It was constructed bycloning 1,683-bp miniplasmid pAO3 (24), an autonomouslyreplicating subfragment of plasmid ColEl (6,646 bp), into theunique EcoRI site of vector XVIII (19). Extracted nucleicacids were hybridized with 32P-labeled single-stranded DNAof recombinant M13 phage harboring various portions ofpAO3 or pAO7 (24) (Fig. 1). Use of single-stranded M13DNA containing appropriate segments derived from ColElDNA as probes enabled us to identify transcript (or nascentDNA strand) polarity and extent. The position of a particularnucleotide on pAO3 was designated by the number ofnucleotides from the ori site, with a plus or minus sign,depending on whether the position was downstream orupstream from the ori site.By using A-type probes (see the legend to Fig. 1), we

analyzed nucleic acids extracted from E. coli K-12 strain 594(Kind) at 20 min postinfection with ASN4 phage at 37°C.Figure 2a shows autoradiographs of M13 DNA fragmentsthat hybridized with nucleic acids as detected by S1 map-ping. From the results, it was possible to deduce the struc-tures of most of the RNA transcripts and nascent DNAchains. When cell extracts were treated with DNase I beforehybridization with M13 probes, the 1,154 (probe 2A)- and663 (probe 4A)-nt bands disappeared but a 436 (probe 5A)-ntband was also visible. These bands were interpreted ashybrids of probes with infecting XSN4 phage DNA, whichcontains the complete DNA of pAO3. The presence andintensity of the 436-nt band were contributed mostly byinfecting DNA but partly by RNA covering the whole regionof probe SA, which extends to + 16. Since we know the inputmolar amount of infecting phage DNA in each sample,estimation of the 1,154- or 663-nt band by hybridization

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1210 INOUE AND UCHIDA

a.1 2 3 4 5 6 7

Probe 5A 4A - 2ADNase -. -+ - +

3001 _

631 ohm

_ parental DNA(1154nt)

.(663nt )

1.........23 4 5Probe 2A 4ARNase - - + +DNase + - -

.. 3

"_eIq- A2 (555nt)-A3 (520nt)

A2-1 (455nt)

396 IW-A2-2 1405nt)

A2-3 (360nt)344

.parentalDNA

A2(555nt

-nascentDNA

-A2-1 (455nt'

b.

Probe 2ARNase + +Acc I +

* -11i54tOO-- _ ,-940

63 -

517506:. .* ,492

396--w.

344-

298-.,a.-344-0298-

A2 (555nt)A2-1 (455nt)A2-2 (405nt)A2-3(360nt)A3 (520nt)

*-- 325

-278

298-4W

221 -4W

.400 200 1 -20 -400 -6600

FIG. 2. Transcription mapping of the wild-type replicon withA-type probes. (a) Si nuclease protection mapping of nucleic acidsextracted from 594(Xind) cells at 20 min postinfection with XSN4phage. In lanes 3, 5, and 7, nucleic acids were treated withRNase-free DNase I. The probes used were 5A (lanes 2 and 3), 4A(lanes 4 and 5), and 2A (lanes 6 and 7). Lane 1 contained 32P-labeled,EcoRI- and Hinfl-digested pBR322 DNA, which served as sizestandards. The numbers to the left of lane 1 indicate the sizes of theDNA standards (numbers of nucleotides). Each RNA transcriptdetected with A-type probes was designated A followed by a serialnumber and the size in nucleotides, e.g., A2 (555 nt) and A2-1 (455nt), etc. The infecting phage DNA containing the complete genomeof pAO3 was detected as 1,154-, 663-, and 436-nt bands with probes2A, 4A, and 5A, respectively. They are referred to as parentalDNAs. (b) Transcription map based on the results of the autoradio-gram. Arrow width indicates the relative amount of each transcript.The name of each transcript and its size as deterrmined by probe 2Aare indicated.

served as the internal control for recovery. With both probes2A (lanes 6 and 7) and 4A (lanes 4 and 5), 555-, 520-, 455-,and 405-nt bands, as well as several shorter bands, weredetected. Chain lengths determihed by different probesindicated that these transcripts lie completely within theregion covered by probe 4A, which contains a fragment ofColEl DNA extending from -652 to +11. Detection of a555-nt band (lanes S and 7) was particularly interesting,because in vitro transcription of the preprimer RNA hasbeen shown to start at -555 (12). Nevertheless, the 455-ntband was the most abundant. With probe SA (lanes 2 and 3),which contains the ColEl region starting at -420 and endingat + 16, 555-, 520-, and 455-nt bands disappeared and,instead, 420-, 405-, and 385-nt bands and several minor oneswere detected. The 420-nt band was the most abundant,indicating that the 455-nt band which was detected by probe2A or 4A corresponds to the 420-nt band detected by probeSA; therefore, the 420-nt transcript detected by probe SAand the 455-nt transcript detected by probes 2A and 4A musthave ended exactly at the ori site. As will be shown later, the

C. Accll -or!

214 278FIG. 3. Mapping of nascent DNA. (a) Si nuclease protection

mapping with probes 2A (lanes 2, 3, and 4) and 4A (lane 5). Nucleicacids extracted from XSN4-infected 594(Aind) cells were treatedwith RNase-free DNase I (lane 2) or heated RNase A (lanes 4 and 5)before Si digestion. Lane 1 contained the 32P-labeled DNA frag-ments that served as size standards. (b) Lanes: 1, size standards; 2,the same sample as in panel a, lane 4; 3, the same sample afterdigestion with AccI. The numbers beside the lanes indicate molec-ular sizes in nucleotides. (c) Schematic representation of the nascentDNA fragment.

455-nt band is a cleaved product of the 555-nt transcriptlacking a 100-nt fragment from the 5' end. The 520-nt banddetected by probes 2A and 4A could be accounted for byassuming a start at position -555 and termination at position-35. These results are schematically summarized as a tran-scription map in Fib. 2b, in which each transcript is named.The 3' ends of more than 95% of the transcripts weremapped at the ori site. A few transcripts, such as the 436-ntband detected by probe SA (lane 3) or the 565-nt banddetected by probe 4A (lane 5), extended beyond the ori site.The molar amount of transcripts reading through the ori sitein a lysogen was roughly estimated by microdensitometryas making up 3% of the total A-type transcripts. Whennonlysogens were used, transcripts starting from outside theColEl DNA and under lambda N control read through theori site, as will be shown later (see Fig. 6). Detection ofreadthrough transcripts provided a control showing that ourmethod can in fact detect RNA endpoints of ColEl tran-scription.When phenol extracts of the phage-infected cells were not

treated with DNase I before hybridization with M13 probes,a 490-nt band was detected by probe 2A but not by probe 4A(Fig. 2a, lanes 4 and 6, and 3a). A 280-nt band from the samesample was detected by using probe 3A (data not shown).

a.

b.

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INITIATION OF ColEl DNA REPLICATION IN E. COLI 1211

Note that the region of ColEl DNA covered by probe 2A or3A but not by probe 4A starts at +492 or at +278, respec-tively, and ends at +11 (Fig. 1). Since both the 490- and280-nt bands disappeared when cell extracts were treatedwith DNaseI, they were the region of ColEl DNA down-stream of the ori site. When they were digested with AccIafter Si treatment, the 1,154- and 490-nt bands disappearedand, instead, 940-, 325-, 280-, and 214-nt bands were ob-served (Fig. 3b). When XSN4 phage DNA extracted fromphage particles was hybridized with probe 2A and treatedwith Si nuclease, only the 1,154-nt band was observed.When the sample was treated additionally with AccI, the1,154-nt band disappeared and, instead, 940-, 325-, and214-nt bands were observed (data not shown). Since there isan AccI site at +278 on pAO3 DNA, the 490-nt band asdetected by probe 2A was identified as the nascent leading-strand DNA of ColEl replication, which started at the orisite and was 492 nt long (Fig. 3c). Bands 940 and 214 nt longwere derived from the 1,154-nt band, whose total length isthe same as that of the ColEl DNA within the 2A probe. The214-nt fragment was also produced from the 492-nt DNA.The 325-nt band was inferred as the parental DNA irregu-larly digested with AccI, because the fragment was alsodetected from EcoRI-digested pAO3 DNA hybridized withprobe 2A, followed by treatment with AccI. The molaramount of the leading-strand DNA synthesized by 20 minafter X phage infection was estimated as about 0.3 to 0.5copy per infecting phage DNA.

Start of most A-type ColEl transcripts synthesized within Xlysogens. We have previously reported isolation of a group ofreplication-defective mini-ColEl mutants called cer (forColEl, replication defective) (21). Preliminary characteriza-tion of some cer mutants has been reported. cer-6 is asingle-base alteration at 160 bp upstream of the ori site, andit replicates in bacteria defective in RNase H activity (20,22). cer-10 and cer-17 are a single-base-pair addition anddeletion, respectively, within an A-T cluster around -575,and they behave as promoter-defective mutations (21). Nu-cleic acids extracted from X-lysogenic cells infected withXSN4cer-10 or XSN4cer-17 were analyzed by Si mapping(Fig. 4). A2 (555 nt), A2-1 (455 nt), and other bands weresubstantially reduced to less than 3% of what we haveobserved with the wild-type ColEl replicon. This confirmedthat they are promoter-defective mutants and suggested thatmost of the ColEl transcripts detected and characterized ashaving the same polarity as the leading strand were undercontrol of one and the same promoter located at -555.

Since the results implied that transcript A2-1 (455 nt) wasa processed subfragment of a longer transcript, A2 (555 nt),we did a kinetic experiment including RNase III-defectivebacterium AB301-105(X) (15). RNase III is an RNase specificto double-stranded RNA and is known to participate in theprocessing of certain species of RNA in E. coli (4, 6, 8). Theresults (Fig. 5) indicate that transcript A2 (555 nt) was muchmore stable in rnc mutant bacteria than in rnc+ bacteria,suggesting that A2 (555 nt) was first cleaved by RNase III invivo to produce A2-1 (455 nt) and other shorter transcripts.Si nuclease protection analyses of nucleic acids extractedfrom cells at various intervals after addition of rifampin tothe culture showed that the half-lives of A2 (555 nt) and A2-1(455 nt) in wild-type bacteria were both about 70 s, whereasthose in rnc mutant bacteria were longer than 5 min and 90 to100 s, respectively (data not shown).A2 (555 nt) was not stabilized in AB301-RNase-19, an

rnc+ strain (5) from which AB301-105 was isolated (data notshown).

1 2 3 4 5 6 7 8 9Probe 2 A 4 Acer + 6 10 17 + 6 10 17

parentalDNA

_ ^ .~631-

517- _" .'506-

396- -

-S.-

U.' -A2-1(455nt)

-A2-2(405nt)

344-

FIG. 4. Transcripts of ColEl replication-defective mutants. S1nuclease protection mapping of nucleic acids extracted from594(Xind) cells infected with XSN4 (lanes 2 and 6), XSN4cer-6 (lanes3 and 7), XSN4cer-10 (lanes 4 and 8), and XSN4cer-17 (lanes 5 and 9).Probes 2A (lanes 2 to 5) and 4A (lanes 6 to 9) were used. Thenumbers to the left indicate molecular sizes in nucleotides.

Mapping of transcripts which start outside ColEl DNA. Adetailed restriction map showed that the direction of tran-scription of the RNA II region within XSN4 phage was thesame as that of the leftward transcription of the early phagegenes which start from the PL promoter (data not shown). Ininfected cells, XN-antiterminated transcription starting at thePL promoter is known to proceed for a substantial distancepenetrating into the b region (25), where the mini-ColElDNA was inserted. Thus, it was possible to test whether atranscript that starts from the upstream PL promoter withinthe vector and transcribes the RNA II region ends at the orisite by infecting a nonlysogen with XSN4. Cell extracts weretreated with DNase I, and transcripts were detected andcharacterized by hybridization with probes 2A, 4A, and 5A(Fig. 6). Compared with those presented in Fig. 2, thesepatterns show a pronounced difference in the presence ofmany bands in Fig. 6 which were longer than A2 (555 nt).Note that we detected only transcripts homologous to ColElDNA. The longest RNA present in lane 1 of Fig. 6 was 1,154nt long, which is the total length of the ColEl DNA presentin probe 2A, including the 492-nt segment downstream of theori site. The band appeared to be 663 nt long when hybrid-ized with probe 4A and 436 nt long with probe 5A because ofthe shorter ColEl DNA present in these probes. Therefore,Al (1,154 nt) was judged to be a readthrough transcriptoriginating from within the A genome, reading the wholeextent of the ColEl DNA fragment present in probe 2A, andagain penetrating the A genome. When this species of RNAwas posttranscriptionally cleaved by RNase III at -455, a950-nt RNA emerged (Fig. 6, lane 1). With probe 4A, theRNA manifested itself as a 465-nt band (Fig. 6, lane 2),which is distinct from the A2-1 (455-nt) band. The Al(1,154-nt) and Al-1 (950-nt) bands, as well as other longer

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1212 INOUE AND UCHIDA

10 1112 13rn c+ rnc_

. ... ....>._S_ _W. _............W - A...................Time: 1 2 3 5 1020 1 2 3 5 1020 (men.)

- parental1000 DNA

631-a

-k- w J -A2 (555nt)*.dw -A3(520nt).~ _ -rscent

-A2-1t455nt)--A2-2(405nt)

396 -_A.0_

4w w.* 4-A2-3(360nt)344-q_ I&

1 2 3Probe 2A 4A 5A

Al (1154nt) _p.-

AI1(95Ont) @

(6r3nt) - 11 631

A 2(555nt)...-517-.506

(465nt)-.A2-'1(455nt)~~~~~~~~~~~~~~~~...

A2 -2 (405nt.) ... 396

298-

123 5 10 20min 123 5 10 20min

FIG. 5. Kinetic analyses of transcripts and nascent DNA. (a) S1nuclease protection mapping with probe 2A. Nucleic acids wereextracted from 594(Aind) cells (lanes 2 to 7) or RNase III-defectiveAB301-105(A) cells (lanes 9 to 14) at 1 (lanes 2 and 9), 2 (lanes 3 and10), 3 (lanes 4 and 11), 5 (lanes 5 and 12), 10 (lanes 6 and 13), and 20(lanes 7 and 14) min postinfection of bacteria with XSN4. Lanes 1and 8 contained size standards. The numbers to the left indicatemolecular sizes in nucleotides. The bands indicated by arrowheadswere derived from bacterial DNA homologous to the M13 sequence,because they were observed when nucleic acids extracted fromuninfected AB301-105(A) cells were treated with RNase A andhybridized with 32P-labeled vector M13mp2 DNA (data not shown).They were also present in A19. One of the bands overlaps with A2-3(360 nt). (b and c) Relative quantities of A2 (555 nt) (closed circle),A2-1 (455 nt) (open circle), and the nascent DNA (open triangle) atthe times indicated were expressed as copy numbers with respect tothe amounts of parental DNA in 594(Xind) (b) and AB301-105(A) (c)infected with XSN4.

bands, were also observed when a nonlysogen was infectedwith XSN4cer-10, which has a defect in the ColEl promoter(data not shown). Therefore, the results (Fig. 6) showed thattranscripts starting outside ColEl DNA read through the orisite.

Analyses of ColEl transcripts and nascent DNA in RNaseH-defective strains. We have reported isolation of RNaseH-defective bacterial mutants that suppress the DNA repli-cation defect of cer-6 mutant plasmids (20, 22). A biochem-

RNA

FIG. 6. Transcription in nonlysogenic cells. Si nuclease protec-tion mapping of nucleic acids extracted from nonlysogenic 594 cellsat 10 min postinfection with XSN4. Nucleic acids were treated withDNase I before digestion with Si nuclease. The probes used were2A (lane 1), 4A (lane 2), and 5A (lane 3). Names of transcripts areshown to the left of the autoradiogram. The sizes of the standardDNA fragments are indicated to the right in nucleotides.

ical assay showed that their RNase H activity was less than0.06% of that produced by the wild-type bacteria (20, 22).ColEl transcripts and nascent DNA were analyzed in thesernh-defective mutants. In SN51 (Xind) (herA39) cells (20)producing thermolabile RNase H, production of ColEl tran-scripts ending at the ori site was the same whether the cellswere incubated at 30 or 42°C (Fig. 7a). Neither a substantialdecrease in the amounts of A2 (555 nt) and A2-1 (455 nt) nora pronounced increase in the amount of transcripts readingthrough the ori site was observed. The 492-nt nascent DNAwas also detected, and its amount stayed almost constant inSN51(Aind), irrespective of the incubation temperature (Fig.7b). Similar results were obtained with SN296(Xind) harbor-ing an amber mutation within the rnh gene (22) and in ON121(kind) and ON152 (Xind) harboring rnh-S9(Ts) and rnh-91(23), respectively (data not shown).Kanaya and Crouch have constructed an RNase H-defec-

tive E. coli strain by insertion of the cat gene into the BamHIsite within the rnh gene (14). Loss ofRNase H activity in thisstrain was considered to be more stringent than that of otherrnh mutants (10). Wild-type ColEl replicated in rnh-339::cat, and the strain suppressed the replication defect of cer-6(data not shown). Therefore, transcripts formed inMIC1020(X) (rnh-339::cat) were analyzed. The amount ofA2-1 (455 nt) was decreased, although the A2 (555-nt) and

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1 2 3 4 5.6 1 2

herA39 her+ rnh rnh339-,(C) ::cat

nparentalr IDNA

" read-through~~~~r-V transcripts

517-506 - "' -A2(555rnt)

040:A2-1396- .. (45&rt)

344-

298-

_nascent - -A2

DNA (555nt)

Is #,- -A2-1

(455nt)

22i1%

RNA DNA DNA

Rf*A

FIG. 7. Transcripts and nascent DNA in RNase H-defective E.coli strains. (a) SN51(Aind) cells harboring a mutant rnh gene

producing temperature-sensitive RNase H were cultured at 30 or

42°C, infected with XSN4, and cultured for 20 min at 30°C (lane 2) or

42°C (lane 3). Nucleic acids extracted and treated with DNase I wereanalyzed with probe 2A. Lane 1 contained the size standards. Sizesare indicated to the left in nucleotides. (b) Si nuclease protectionmapping of DNA extracted from SN51(Xind) cells cultured at 32°C(lane 2), 37°C (lane 3), and 42°C (lane 4). SN50(Xind) (lane 5) and594(Xind) (lane 6) cells were cultured at 37°C and infected withXSN4. Probe 2A was used. (c) S1 nuclease protection mapping ofnucleic acids extracted from MIC1001(A) (rnh+) (lane 1) andMIC1020(A) (rnh-339::cat) (lane 2) cells infected with XSN4.

A2-1 (455-nt) bands were still detectable (Fig. 7c). Longertranscripts which extend beyond the ori site were definitelypresent (Fig. 7c). Microdensitometry of autoradiographicbands hybridizing with probe 4A showed that about 65% ofthe transcripts, starting from -555, read through the ori site(data not shown).

DISCUSSION

The results obtained by analyses ofRNA transcription andsynthesis of nascent DNA in rnh+ bacteria infected withX-mini-ColEl hybrid phages can be summarized as follows.(i) Synthesis of RNA transcripts on leading-strand ColElDNA starts at -555, and that of most of them ends at the orisite. Transcripts extending beyond the ori site, however,were estimated to be about 3% of the total leading-strandtranscripts (Fig. 2). Although shorter RNA fragments, suchas the 455-nt fragment, were detected, they were mostlydegradation products of 555-nt RNAs because these RNAs,as well as the 555-nt RNA, disappeared in two ColElreplication-defective mutants which have mutations in thepromoter for 555-nt RNA (Fig. 4). The transcripts areprocessed into several shorter fragments by at least RNaseIII (Fig. 5), and processing by RNase III can be the first step

in degradation of the RNA transcript in vivo. Synthesis ofnascent ColEl DNA starts at the ori site, in qualitativeagreement with the results of in vitro studies (12). (ii) Themolar ratio of the nascent DNA starting at the ori site to the555-nt RNA ending at the ori site was rather small. Theinitial rate of synthesis of the leading-strand transcripts was0.5 to 1.2 copies per infecting X-mini-ColEl phage DNA permin, whereas the rate of nascent DNA synthesis was esti-mated as about 0.04 copy per infecting X-mini-ColE1 phageDNA per min in rnc mutant bacteria infected with the phageat multiplicities of infection of around 5 (Fig. 5). Theseobservations, if taken at face value, were in quantitativedisagreement with the in vitro finding that the preprimerRNA starting at -555 is cleaved by RNase H at the ori siteto initiate DNA synthesis with a switching frequency of atleast 60% (26). In vitro studies showed that a large fraction ofthe ColEl primer precursor transcripts form an extensivestable hybrid with the template DNA, beginning near thereplication origin and extending downstream (26). The samemay be true in vivo. However, after cleavage with RNase Hat the ori site, most of the upstream portion of the cleavedtranscripts stay only transiently on the template DNA,especially in vivo, and leave the template DNA before DNApolymerase I can use the RNA as the primer for initiation ofDNA synthesis. Thus, the present study indicated that, perse, cleavage of the RNA at the ori site in vivo did not alwayslead to initiation of DNA synthesis from the site.We have previously isolated bacterial suppressors called

herB that allow replication-defective cer-6 ColEl DNA toreplicate in rnh+ bacteria (22). The suppressor mutationmapped within or close to the polA gene, and substantialincreases of DNA polymerase I activity in extracts of herBsuppressor mutant cells were observed. Therefore, overpro-duction or structural alteration ofDNA polymerase I in herBmutant cells was suggested to explain suppression of thereplication defect of cer-6 mutant ColEl (22). Furthermore,we have reported isolation of bacterial mutants which sup-press the replication defect of cer-17 mutant ColEl (20).cer-17 maps within the promoter for RNA II, and infection oflysogenic bacteria with the X-mini-ColE1 cer-17 hybridphage produced less than 3% of leading-strand transcriptsthat were produced by phages having the wild-type ColElreplicon (Fig. 4). One possibility would be to assume that thesuppressor mutants utilize the reduced transcripts of thecer-17 mutant more efficiently by alteration of an unidenti-fied gene product of the cell to initiate ColEl DNA synthe-sis. Further studies on these bacterial mutants are necessaryto clarify which accessory genes are involved and detail thesteps that control the efficiency of initiation of ColEl DNAsynthesis in vivo.On the basis of analyses of several rnh mutants, we

concluded that production of the transcripts ending at the orisite in vivo is due mostly to cleavage of the transcripts at theori site by RNase H activity. In fact, it was observed that thenumber of transcripts ending at the ori site was muchreduced and that of the readthrough transcripts was substan-tially increased in the rnh-339::cat strain (Fig. 7c). In con-trast, experiments using rnh mutations other thanrnh-339::cat indicated cleavage of the RNA at the ori site,and we failed to observe a substantial fraction of transcriptsreading through the ori site. We conclude that the rnh ts andam mutations used were leaky and cleavage of RNA at oriwas due to the action of the residual RNase H activities,although these were far below the detection limit of thebiochemical assays (20, 22).We have reported previously that cells harboring defects

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1214 INOUE AND UCHIDA

in the rnh gene suppress replication of a replication-defectiveColEl mutant sustaining a mutation called cer-6 (20). Thepresent results indicated that these rnh mutant cells musthave retained residual RNase H activities. On the otherhand, cer-6 mutant ColEl also replicates in rnh-339::catmutant cells. Therefore, the question of why the presence ofthe wild-type level of RNase H activity was detrimental toDNA replication of cer-6 mutant ColEl but mutant levelswere not remains. We have previously postulated that thecer-6 mutation may have introduced a new site sensitive towild-type RNase H activity by affecting the conformation ofat least part of the primer RNA to become available forhybridization with DNA (21). It is also possible that theRNA-DNA hybrid of cleaved cer-6 RNA is extremely un-stable and is marginally able to function as a primer. Thus,cer-6 mutant DNA may replicate in vivo if either the RNaseH activity is lowered by a defective mutation or the DNApolymerase I activity is elevated by herB mutations. In themost defective rnh mutant cells (rnh-339::cat), ColEl DNAmay replicate, for example, by the type III mechanismpostulated by Dasgupta et al. (3). For the mechanism tooperate, RNase H activity is not only dispensable butprobably inhibitory. Our data show that 30 to 40% of thetranscripts still ended at or near the ori site in thernh-339::cat strain (Fig. 7c). It is possible to interpret thefinding as showing that the cell still has residual RNase Hactivity. In fact, Itaya has recently isolated a second RNaseH encoded by the rnhB gene of E. coli (9). If the secondRNase H can cleave the ColEl transcripts as the primaryRNase H does, and if we can demonstrate that DNAsynthesis initiates at the ori site in rnh-339::cat cells, thenthe classical type I mechanism is still a valid model toexplain ColEl DNA synthesis in rnh-339::cat cells.

ACKNOWLEDGMENTS

We thank I. Saito and H. Handa for advice on Si nucleaseprotection mapping and R. J. Crouch for providing strain MIC1020(rnh-339::cat) and unpublished results of his group. We also thank S.Naito for reading the manuscript and K. Kawakami and M. Itaya forvaluable discussions.

This work was supported in part by a grant from the Ministry ofEducation, Science and Culture of the Japanese Government.

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