Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase

J. Mol. Biol. (1978) 124, 373-389

Discontinuous Replication of Colicin El Plasmid DNA in a Cell Extract Containing Thermolabile DNA Ligase YOSHJMASASAKAKIBARA

Department of Chemistry, National Institute of Health Z-10-35, Kamiosaki, Shinagawa, Tokyo 141, Japan (Received 23 February 1978, and in revised form

12 June 1978)

A cell extract prepared from the Zig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin El plasmid at 25°C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from t,he thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25°C to 32”C, whereas that in the Zig+ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the Zig-ts extract, even at 25”‘C. The DNA pulse-labelled with the Zig& extract for 30 seconds at 32°C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular DNA-DNA hybridization experimolecules upon chasing with a Zig+ extract. ments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5’-to-3’ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component. The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plssmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compoumd does not interfere with elongation of progeny strands. Chain elongation in the Zig-ts extract at 25°C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the Zig& extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin El plasmid DNA replicate discontinuously.

1. Introduction For

elongation

(1968,1973) by joining

have

of

DNA

proposed

of the DNA

strands

in

a mechanism

chains

by DNA

semiconservative of synthesis ligase.

replication, of short

The finding

DNA

of transient

Okezaki et al. chains

followed

accumulation

DNA chains in the progress of replication has shown that the discontinuous mechanism operates in at least one of the two strands, which elongates in the direcOion opposite to the movement of the replication fork. For studying whether only one strand or both strands elongate discontinuously, the small duplex DNA molecule of colicin El plasmid, which replicates unidirectionally from a fixed region (Tomizawa

of short

373 0022-2830/78/260373-17 %02.00/O

0 1978 Academic

Press Ino. (London)

Ltd.

371

\‘.

tiAKAKIB>\RA4

ct al., 1974: lnselburg,

1974; Lovett~ et al., 1974). provides a convrnifmt system. ‘I’hv rcplicative intermediates of the plasmid DNA usually have a double-stranded replicating loop. Transient accumulation of short, DNA fragments of the plasmid DNA has been observed in chloramphenicol-t,reated colicinogenic bact)eria, (Inselburp $ Oka, 1975), and in the plasmolysed cells (Staudenbauer. 1974). ln the experiments described in this paper? we have examined in vitro replication of Co1 Elt DNA in a cell ext’ract from Escherichia coli wit’h bhe temperat’ure-sensit,ive mutation Zig-k?‘. which produces t,hermolabile DNA ligase (Pauling & Hamm, 1968; Gottesman et al., 1973; Konrad et al., 1973). Co1 El DNA molecules replicating in an extract deficient, in E. coli DKA ligase were found to contain short DNA fragments on hot’h strands. Recently, E. coli mutants with reduced dUTPase activity have been shown t,o accumulate bransiently short DNA fragments, probably as a consequence of incorporation of uracil into DNA (Tye et al., 1977). We have also examined whether t,hr short DNA fragments found in the in vitro replication system had been generated as a result of incomplete repair of misincorporated nucleotides. Although the cell extract used for the plasmid DNA replication was able to repair Co1 El DNA containing apurinic sites, the joining activity of E. coli DEA ligase for completion of t,he repair was blocked by nicotinamide mononucleotide, which does not interfere wit’h elongalion of progeny strands in the replication system (Sakakibara & Tomizawa, 1974c). On the basis of these results, we propose in this paper discontinuous replication of both strands of Co1 El DNA. We furt,her discuss the function of E. coli DNA ligase in the plasmid DNA replication.

2. Materials and Methods (a) DNA synthesis

in

cell extracts

A cell extract containing thermolabile DNA ligase was prepared from E. co/i NT491 The thr Zefr thi str m&A endA Zig-ts7, which was provided by M. Gellert and J. Tomizawa. strain was constructed by transduction with Pl phagr grown on Zig-ts7 pts + bacteria from a YSl pts strain, which was made by mating Hfr CHE-11 pts (Epstein et al., 1970) and YSI (Tomizawa et al., 1975). TI me cells of NT491 xvere grown t,o approx. 2 x IO8 cells/ml at 30°C in a Casamino acids medium (Sakakibara S: Tomizawa, 1974a) supplemented with 0.25% Bacto-peptone (Difco) and 0.050,. veast extract (Difco), followed by further cultivation for 2.5 h in the presence of chloramphenicol (180 &ml). Cell extracts were prepared 1974a). The extracts from the lig-ts as described previously (Sakakibara 8r Tomizawa, cells contained a significant amount of cellular DNA of small molecular weight, which was not found in extracts from wild-type cells. The contaminating cellular DNA was digested by incubation of the extracts at 25°C for 30 min. This treatment did not significantly affect the activity of extracts for synthesis of Co1 El DNA. Cell extra&s from YS17, a t,hermoresistant revertant of NT491, were prepared in the same way. Wild-type cell extracts were prepared from YS 1 cells cultured in the above medium at 37°C and then in the presence of chloramphenicol (180 &ml) for an additional 2 II, and used for assay without preincubation. The standard reaction mixture (300 ~1) contained 20 rnnI-potassium 0.2 rn.n-NAD, 0.2 Inn% of each of phosphate buffer (pH 7.4), 50 m;n-KCl, 7.5 mm-MgCl,, t,he 4 ribonucleoside triphosphates, 0.025 II~M of each of the 4 deoxyribonucleoside triphosphates (dNTPs), 120 ~1 of a cell extract and 3 pi of Co1 El DNA. Co1 El DNA templates were prepared as described previously (Tomlzawa et al., 1975). Incubation was performed at 25°C or 32°C. Unless otherwise noted, the specific activities of [c(-32P]dTTP and [methyl-3H]dTTP were approx. 1 Ci/mmol and 20 Ci/mmol, respectively. The methods for termination of thr rract,iorr and for extraction of DNA were as drscribed previously t Abbreviations used: Co1 El, colicin El plasmid; CC molecule, meric molecule ; OC molecule, open circular monomeric molecule.

closed circular

twisted

mono-

DISCONTINUOUS

REPLICATION

OF COL

El

l)Nd

375

(Sakakibara & Tomizawa, 1974a). The extracted DNA was dialysed against 50 111MTris.HCl buffer (pH 8.0) containing 50 mm-K,HPO,, 50 mM-NaCl, 5 mM-EDTA and 0.25% sodium N-lauroyl sarcosinate. Sources of the materials used have been previously described (Sakakibara & Tomizawa, 1974a,b,c). 14C-labelled CC molecules of Co1 El DNA containing apurinic sites (approx. 2 sites per molecule), which were used for studies of repair of damaged DNA in cell extracts, wet-c’ gifts from T. Inoue and T. Kada. The DNA was prepared by incubation of CC molecules labelled with [14C]thymine (approx. 6.0 x lo3 cts/min prr pg) at, 70°C for 30 min in a mixture of 10 mw-sodium citrate and 0.1 M-NaCl, which was adjusted to pH 5.0 with 1 N-HCl (Verly & Rassart. 1975). Phage T4 DNA ligase was purchased from Miles Laboratories. (b) Sedimentation

analysis

Sucrose solutions were made up in 0.15 M-NaCl, 0.015 M-sodium citrate and 5 mM-EDTA or in 0.3 M-NaOH, 1.0 M-NaCl and 5 mM-EDTA. Samples (0.1 to 0.15 ml) were layered on was performed at 4.7 ml linear gradients of 5% to 20% (w/v) sucrose. Centrifugation 45,000 revs/min for 140 min at 10°C in neutral gradients. and for 115 min or 6.5 h at 5°C in alkaline gradients, using a Beckman SW50.1 rotor. Samples were taken from the bottoms of the tubes. The reference Co1 El DNA was prepared from A745 thy (Co1 El) bacteria labelled with approx. 30 mCi [3H]thymine/mmol or IO mCi ~‘4C]thymine/mmol as described previously (Sakakibara & Tomizawa, 1974a). The reference DNA in neutral gradients was OC and CC molecules, and that in alkaline gradient#s was single-stranded linear molecules of unit length and collapsed CC molecules. Linear molecules of Co1 E 1 DNA were prepared by digestion of CC molecules with the restriction endonuclease EcoRI as described previously (Tomizawa et al., 1974). EcoRI was prepared from E. coli B end (R,) according t,o the method described by Yoshimori (1971). (c) DLVA-DNA

h~ybridization

The separated strands of Co1 El DNA were prepart,d from denatured linear molecules by CsCl density gradient centrifugation in the presencc~ of poly(U,G) (Sakakibara & Tomizawa, 1974c) The Hue11 fragments of Co1 El DNA were gifts from M. Takanami. Hybridization was performed essentially according to t)lle m&hod described by Denhardt (1966). The amount of DNA immobilized on a nitrocellulose filter disc (Millipore. HA-O.45 pm) was 0.5 pg each for the light (I,) and heavy (H) strands, and 1.35, 1.0. 0.65, 0.4, 0.2 and 4.0 pg for the HaeII A to E fragment>s and Co1 El DNA, respectively. HybridizaGon was carried out at 65°C for 20 h in a solution of 0.45 fir-NaCl and 0.045 Xsodium citrate (pH 7.0) with duplicate samples. After iucubation the filter discs were washed with 3 ml\l-Tris.HCl buffer (pH 9.4) corltaining 2 rnnz-EDTA. Elution of hybridized DNA from each filter disc was performed t),v heating at 95°C for 10 min ill 0.5 ml of 10 mM-TriseHCl buffer (pH 8.0) containing 0. I rnM-EDTA aud 10 pg of denatured calf thymus DNA. Recoveries of labelled DNA were more t’hau !)OO,<,.The amount of DNA hybridized was determined as the average of each duplicatcl set. Blank values subtracted were radioactivities trapped to filter discs wit,h 0.5 pg calf thymus DNA, which were less than 0.57’ of input radioactivities. Purity of the separatcid st,rands and the HaeII frapments used was examined by hybridization against labelled L and H DNA and against labelled HaeII C, D and E fragments, respectively, whicbh were prepared analytically from Co1 El DNA labelled with approx. 30 mCi [3H]tt~~-mir~r~/rnmol. The amounts of eacll labelled HaeII fragment trapped t’o filter discs wit11 diff’ererlt classes of HaeII fragments were the level of blank values, when t,hose with the, corresponding fragment, WIVV 300 to 500 cts/min. The amount of labelled L DNA trapped to a filt,er disc with the L strand ant1 lahelled H DNA trapped to that with the H strand was 35 cts/min and less than 10 cts/min, respectively, under the conditions where the amount of labelled L and H DNA hybridized to the complementary strand was 345 and 360 cts/min. respectively. In experiments for analysis of strand component)s t,hr fraction of tile H-st,rantl compotlrrlt may be slightl) overestimated. DNA samples for hybridization were prepared as follows. Pulse-labelled DNA was fractionated by alkaline sucrose gradient, centrifugation and the fractions were separately pooled according to the size of labelled DNA as described later. To the pooled fractions

Y. SAKAKIBAHA

376

50 pg of denatured calf tltylrxus DNA WRSadded at~d th Inixtllrc: was Ilc~utrttlieed witAl 3 N-HCl. After additiurl of 2 vol. prechilled ctllanol, ttlc: ruixt,llrc:s \vorc~k(spt, at. 20°C: for 60 min and centrifuged at 4°C for and heittecl 0.3 ml of 0.5 M-N&H cular weight of the DNA, followed 1968). Recoveries of labelled DNA mixture from the 7 S fractiorl was

and fragmentation

20 rrlin at. 3400 rt:vs/min. ‘I’tlf. p~:llc~~s w(‘rc! dissolvc~cl ill for 5 Inill in a boiling water bath to reducr~ ttlc% IIIOIPby neutralizatior~ wit11 1 N-HCI (‘l’olnie>Lwa & C)g;twa, were more than 8596. III some cxxperiments, ttlt: DNA used for hybridization witllout, rttlnnol precipitation

by alkaline trcatrnent.

3. Results (a) Synthesis of Co1 El DNA

in un extract from the lig-ts cells

An extract from the Zig-ts7 cells was incubated with Co1 El DNA in the standard reaction mixture at 25°C and at 32°C. As shown in Figure l(a), the amount of dTMP incorporated into acid-insoluble material was much smaller at t,he higher temperature than at the lower temperature. This lowering of the incorporation at the higher temperature was not observed with an extract from the thermoresistant revertant cells (Fig. l(b)). Co1 El DNA synthesis in the Zig-k extract was also examined in the presence of 10% glycerol and 2 mM-spermidine. The addition of these compounds has been shown to lead to the accumulation of early replicative intermediates containing newly synthesized 6 S DNA fragments (Sakakibara & Tomizawa, 19743). The amount of incorporation in the presence of glycerol and spermidine was not significantly affected by the elevation of t,emperature from 25°C to 32°C either with the Zig-ts extract (Fig. l(a)), with the Zig-ts extract. or with the Zig+ revertant extract (Fig. l(b)). Th e incorporation

incubation

time (rnirl) (b)

FIG. 1. Co1 El DNA synthesis with cell extracts from a Zig-t8 strain and its revertant at low and high temperatures. The extracts from NT491 Zig-t87 cells (a), and from cells of Y817 (a thermoresistant revertant of NT491) (b), were incubated at 25°C and at 32°C under the standard assay conditions, or with 10% (v/v) glycerol and 2 mM-spermidine added. The specific activity of [a-3aP]dTTP was 200 cts/min per pmol. A 20 ~1 sample was withdrawn from each reaction mixture at the times indicated and the radioactivity of the acid-insoluble fraction was measured. The effect of addition of rifampicin (10 pggiml) at the start of incubation was also examined. -O-O--, Standard conditions at 25°C; --O--O--, standard conditions at 32°C; -e-e-, + glycerol + spermidine at 25OC; --@--a--, + glycerol + spermidine at 32°C; -U-o--, + rifampicin at 26°C; --w--m-, + glycerol + spermidine + rifampicin at 25°C.

DISCONTINUOUS

REPLICATION

OF

COL

El

DNA

377

in the presence of glycerol and spermidine as well as in their absence, was completely blocked by rifampicin added at the start of incubat’ion (Fig. l(a)). Rifampicin inhibits initiat,ion of Co1 El DNA replication (Sakakibara & Tomizawa, 1974a). DNA formed in the presence of glycerol and spermidine with the Zig-ts extract during incubation for 60 minutes at 25°C as well as at 32°C consisted exclusively of molecules containing 6 S DNA fragments, and they sedimented at a position roughly corresponding to 26 S in a neutral sucrose gradient (data not shown), as did the early replicative intermediates formed with extracts from YSlO(Co1 El) and YSl cells (Sakakibara & Tomizawa, 1974h; Tomizawa et al., 1975). These results show that the reduction of incorporation at the higher temperature with the lig-ts extract was not due to inhibition of synthesis of early replicative intermediates. The effect of higher temperature on the further progress of replication from early replicat,ive intermediates to completion of replication was next examined. In this experiment rifampicin was used to block further initiation of Co1 El DNA replication without interfering with completion of replication once it was initiated (Sakakibara & Tomizawa, 1974a). The standard reaction mixture containing the lig-ts extract was incubat-rd at 25°C and rifampicin was added 15 minutes after the start of incubation, followed by further incubation at 25°C for 15 minutes. A portion of the reaction mixture was then transferred to 32°C; the remainder was incubat’ed further at 25°C. As shown in Figure 2(a), the amount of incorporation at the low temperature increased linearly in the presence of rifampicin, but the increase ceased upon transfer to the high temperat’uro. Thus it appears that elongation of progeny &rands from early replicative intermediates t,o the completion of replicat,ion was temperature-sensitive in the lig-ts extract. The amount of incorporation with the Zig-ts extract was greatly reduced at 32”C, but the rate of incorporation at this temperature, as measured by pulse-labelling for three minutes, was more than 30% of that at 25°C even after 120 minutes of incubation (Fig. 2(b)). The increase of the amount of incorporation in the rifampicintreated reaction mixture after transfer from 25°C to 32°C had almost ceased within 30 minutes of incubation at the high temperature (Fig. 2(a)). The rate of incorporation in the rifampicin-treated reaction mixture at 30 minutes after the transfer, however, after the was more than 80% of that at 25°C (Fig. 2(b)). Th e rate of incorporation transfer decreased gradually during prolonged incubation. The cessation of incorporation upon continuous labelling at the high temperature with the Eig-ts extract seems likely to be a result of equilibrium between synthesis and degradation. Since 3Hlabelled CC molecules added to the reaction mixture containing t’he lig-ts extract and rifampicin remained closed circular, without significant loss of label, even after 60 minut!es of incubation at 32°C (data not shown), the molecules replicated at the high temperat’ure in the Zig-h extract appear to have a peculiar structure so that t,hey were subject t’o some nuclease attacks. (b) Acc~tnulation

of newly formed

open

circular

monomeric

molecules

The DNA labelled with the Zig-ts extract at 25°C for 60 minutes was analysed by neutral and alkaline sucrose gradient centrifugation. As shown in Figure 3(a), the labelled DNA con&ed of two major components in a neutral sucrose gradient; one sedimented with the reference OC molecules, and the other at a slightly faster rate than t-hc reference CC moleculrs. Tn an alkaline sucrose gradient, the labelled DNA separated into fragments of approximately 6 S, single-stranded linear molecules of

30

90

60 (a)

120 lncubatlon ilme (mln)

I

I

/

1

30

60

90

120

(b)

FIG. 2. Analysis of Co1 El DNA synthesis wit,h the Zig-h extract at low and high temperatures by continuous and pulse-labelling. Four samples of the standard reaction mixture containing the Zig-ts extract and [a-s2P]dTTP (140 cts/ min per pmol) were provided. Two samples of each were incubated at 25°C and at 32°C throughout the experiment. The other 2 samples were incubated at 25°C and rifampicin (10 pg/ml) was added 15 min after the start of incubation, followed by further incubation at 25°C for 15 min. One of the samples supplemented with rifampicin was then transferred to 32°C; another sample was incubated further at 25°C. For pulse-labelling, [3H]dTTP (approx. 6 Ci/mmol) was added to 45 ~1 of each reaction mixtures withdrawn at the times indicated, and incubation was continued for an additional 3 min. The reaction was terminated by thn addition of a mixture of 10% trichloroacetic acid and 0.1 >z-sodium pyrophosphatr. The (a) 32P and (b) 3H radioactivities of the acid-insoluble fract,iou were measured. ~o~<~--, At 25°C; --O----(1 ---, at 32°C; -e-e-, at 25°C’ with rifampicin; -- l -- l --, at 25°C with rifampicin followed by transfer to 32°C.

unit length, and a small amount of collapsed CC molecules (Fig. 3(b)). These results show that the bulk of the labelled DNA was composed of OC molecules and molecules containing newly synthesized 6 S DNA fragments. The latter molecules presumably corresponded to early replicative intermediates. The product of replication with the Zig-&s extract contained five times more OC molecules than CC molecules. By contrast, the amount of OC molecules in the product of a parallel incubation wit$h the Zig+ revertant extract was approximately half that of CC molecules (data not shown). The OC molecules formed with the Zig-ts extract, as isolated from the neutral sucrose gradient (Fig. 3(a)), contained labelled single-stranded linear DNA of unit, length, and the linear DNA had equal amounts of radioactivity in the light (L) and heavy (H) strands, when these were separated by CsCl density gradient, centrifugation in the presence of poly(U,G) (data not shown). When the OC molecules were treated with the endonuclease EcoRI, which introduces a unique double-stranded break in Co1 El DNA molecules (Tomizawa et al., 1974; Inselburg, 1974; Lovett et al., 1974), the labelled linear strands of the EcoRI-treated molecules separated into fragments of two distinct size classes in an alkaline sucrose gradient, as shown in Figure 4. The lengths of the two fragment)s were calculated to be approximately 200/, and 80% that’ of a single-stranded DNA molecule of unit length. These results indicate that the newly synthesized L and H strands of the OC molecules had an interruption located

DISCONTINUOUS

&

REPLICBTION

OF COL El

DNA

6

$ x G z \ 5 a 3 w

4

2

IO

20

30 Fmctlon

number (bl

(a)

FIG. 3. Neutral and alkaline sucrose gradient centrifugation of DNA synthesized with the Zig-& ext,ract at 25°C. The standard ma&ion mixt,ure containing the Zig-la extract and [a-32P]dTTP was incubated at 25°C for 60 min. DNA was extracted and analysed by neutral (a) and alkaline (b) sucrose gradient centrifugation. The reference DNA in (a) was, from the left, CC and OC molecules, and t,hat in (b) collapsed CC molecules and single-stranded linear molecules of unit length. In this and the following Figures, numbers on the left of each panel are for 32P and t,hose on the right for %. ---;j -~-3--, [32P]DNA; (-----) reference [3H]DNA.

2076 from the cleavage site of EcoRT. OC molecules containing such in the newly synthesized strand have been found in DKA pulselabelled wit*h wild-type cell ext,racts (Sakakibara & Tomizawa, 1974c). They are formed at separation of daughter molecules and are the precursors of CC molecules (Sakakibara et al., 1976). The above result, therefore. implies that replication of Co1 El DNA in the lig-ts extract proceeded unidirectionally. as in wild-type cell extracts (Tomizawa ef al., 1974). Most of the OC molecules formed with the Zig-k extract were converted to CC molecules upon further incubation with the Zig-ts extract at 25°C for 60 minutes in

a,pproximatelv

an interruption

Frccton

number

FIR. 4. Alkaline sucrose gradient centrifugation of OC molecules formed with the Zig-tsextract after treatment with EcoRI. OC molecules were isolated from DNA labelled ait,h the Zig& extract at 25°C for 60 min by neutral sucrose gradient centrifugation (Fig. 3(a)). The OC molecules were treated with EcoRI to introduce a unique double-stranded break, and were analysed by alkaline sucrose gradient centrifugat,ion. The reference DNB was single-stranded linear molecules of unit length. -g-o--, [3aP]DNA; (-----) reference [3H]DNA.

380

Y.

SAK.1

KlHAH;\

t hc presence of rifampicin (data not shown). ‘I’twreforf~, the Ziy-ts extract was less act’ivs in closure of the progeny strands of newly formed OC molecules than big+ extract’s even at the low temperature. The extract of the Zig&7 cells is known to havr a reduced DNA ligase activity even at 25°C (Konrad et al., 1973; Gottesman et al., 1973). It has been shown that closure of the newly synthesized st,rands of daughter OC molecules is blocked by nicotinamide mononucleotide (Sakakibara & Tomizawa, 1974c), which is an inhibitor of E. coli DNA ligase (Gellerb et al., 1968). The present results provide more direct evidence that E. coli DNA ligase is involved in closure of the newly synthesized strands of daughter OC molecules. (c) Retardation

of elongation of progemy strands

The DNA product made with the Zig-h extract at 25°C was pulse-labelled for 12 minutes from the 60th minute of incubation. The labelled DNA consisted of OC molecules and molecules sedimenting faster than the reference CC molecules in a neutral sucrose gradient, as shown in Figure 5(a). Tn an alkaline sucrose gradient, the labelled DNA separated into single-stranded linear molecules of unit length and molecules of various sizes shorter than unit length (Fig. 5(b)). When the DNA sample was briefly treated with DNAase 1 so that closed circular molecules were converted to open circular molecules, most of the labelled DNA sedimented in the fractions between the reference species of OC and CC molecules in a neut)ral sucrose gradient (data not shown). These results show that t,he bulk of the labelled molecules had intact parental strands and contained newly synthesized DNA of various lengths. The pulse-labelling for 12 minutes with the Zig-h extract at 25°C was followed by dilution with 20-fold amounts of a reaction mixture containing the lig-ts extract, an excess amount of unlabelled dNTPs and rifampicin, and then by subsequent incubation at 25°C for 60 minut’es. As shown in Figure 6, the fraction of molecules COIItaining labelled DNA of various sizes was greatly reduced and, inst’ead, t’he fraction of CC molecules was increased without significant loss of the labelled DNA. This result indicates that the molecules containing newly synthesized DNA of various

6

Froctlon number (a)

(b)

FIa. 5. Neutral and alkaline sucrose gradient centrifugation of DNA pulse-labelled for 12 min at 25°C with the Zig-b extract. DNA product made with the Zig-ts extract at 25°C under the standard assay conditions was pulse-labelled for 12 min from the 60th min of incubation with [cr-32P]dTTP, and was analysed by n+eutral (a) and alkaline (b) sucrose gradient centrifugation. -O-O-, [3ZP]DNA; (-----) reference [3H]DNA.

DISCONTINUOUS

REPLICATION

OF

COL

El

DNA

381

Frcctlon number FIG. 6. Alkaline sucrose gradient centrifugation of DNA pulse-labelled for 12 min at 25°C with the Zig-ts extract before aud after chasing with the Zig-& extract,. The standard reaction mixture was incubated at 25°C for 60 ruin without labelled dTTP. At that containing the Zig& extract time [a-32P]dTTP (approx. 10 Ci/mmol) was added and incubation was cont,inued for an additional 12 min. Then a 20 ~1 sample of the reaction mixture was poured into 400 ~1 of a prewarmed 0.26 mnr-unlabelled dNTPs and rifampiciu lOpg/ml, reaction mixture containing the Zig-b extract, and incubation was continued at 25°C for an additional 60 min. The acid-insoluble radioactivities of the reaction mixtures before and after chasing were not significantly different. DNA was extracted from the reaction mixtures, and was analysed by alkaline sucrose gradient centrifugation. Two centrifugation patterns are superimposed in a single panel for comparison. The ordinat,e shows the peroentage of the total radioactivity (approx. 6000 cts/min) recovered in each fraction. :1--o--, Before chasing; -- l .-- l --, after chasing.

lengths were replicative intermediates which had undergone various extents of replication. When the DNA product made with the Zig+ revertant extract was pulse-labelled for 12 minutes from the 60th minute of incubation at 25”C, the labelled DNA consist,ed of approximately 60% monomeric molecules of OC and CC and 40% replicative intermediates, and more than 80% of the label in replicative intermediates was found in 6 S DNA fragments derived from early replicative intermediates (data not shown). Thus the proportion of replicative intermediates containing DNA chains of various lengths in pulse-labelled DNA was much smaller with the Zig+ revertant extract than with the Zig-ts extract. For labelling of molecules replicating to various extents with Zig+ extracts in a similar proportion to that in the DNA labelled with the Zig-ts extract (Fig. 5), pulse-labelling for less than two minutes, instead of 12 minutes, at 25°C was necessary. These results show that the apparent rate of elongation of progeny strands in the Zig-ts extract was much smaller than that in Zig+ extracts. (d) Transient

accunmZution

of short DNA

fragmen,t.s

To analyse the structure of the progeny strands of molecules replicating in the kg-ts extract, the strands were briefly pulse-labelled under the following conditions. The standard reaction mixture containing the Zig-ts extract was incubated at 25°C and rifampicin was added 15 minutes after the start, of incubation, followed by further incubation at 25°C for 15 minutes. A portion of the reaction mixture was then transferred to 32°C; the remainder was incubated furt,her at 25°C. Pulse-labelling with

3x2

Y.

SAKAKIHARA

13H]dTTP at the higher temperature was performed for 30 seconds from the 20th minute after the transfer. Pulse-labelling at, the lower temperature was performed for 30 seconds from the 35th minute after the addition of rifampicin. The major components of the DNA labelled at the low temperature were replicating molecules which sedimented faster than CC molecules in a neutral sucrose gradient (Fig. 7(a)). The DNA labelled at the high temperature behaved similarly in the gradient (Fig. 7(c)). showing that the bulk of the molecules replicating at the high temperature also had intact template strands. In an alkaline sucrose gradient, the DNA pulse-labelled at the high temperature as well as at the low temperature gave a peak of approximately 7 S, in addition to the molecules of various sizes, as shown in Figure 7(b) and (d). Upon chasing of the DNA pulse-labelled at the high temperature with a Zig+ extract in the presence of rifampicin at 32°C for 15 minutes, more than 709/, of the labelled

Fractm

number

FIG. 7. Neutral and alkaline sucrose gradient centrifugation of DNA pulse-labelled for 30 s at 25°C and at 32°C with the Zig& extract. The standard reaction mixture containing the Zig-t8 extract was incubated at 26°C without labelled dTTP. Rifampicin (10 pg/ml) was added 15 min after the start of incubation, and incubation was continued at 25°C for an additional 35 min. At that time [3H]dTTP was added and the reaction wa8 terminated after 30 8 ((a) and (b)). For preparation of a DNA sample pulse-labelled at 32’C for 30 s ((0) and (d)), the standard reaction mixture supplemented with rifampicin at 16 min after the start of incubation at 25°C was transferred to 32°C after 16 min of the addition of rifampicin, and [3H]dTTP was added at the 20th min after the transfer. DNA was extracted and analysed by neutral ((a) and (0)) and alkaline ((b) 14C-labelled linear Co1 El DNA. and (d)) sucrose gradient centrifugation with the reference Numbers on the left of each panel are for 3H and those on the right for 14C. -O-O-, [3H]DNA; (-----) reference [lW]DNA.

I)ISCONTINUOUR

REPLICATION

OF

COL

Kl

DNA1

383

DNA was found in the fractions of OC and CC molecules (data not shown). Labelled DNA sedimenting in the region between the reference species of OC and CC molecules in the neutral sucrose gradients (Fig. 7(a) and (c)) consisted mostly of open circular molecules containing labelled single-stranded DNA4 of various lengths shorter than unit length (data not shown). To examine whether the 7 S DNA fragments were derived from both strands of molecules replicated to various extents, the DNA pulse-labelled at the high temperature was fractionated by alkaline sucrose gradient’ centrifugation (Fig. 7(d)), and the DNA in the 7 S fraction was characterized by the following DNA-DNA hybridization experiments. The isolated 7 S DNA was subjected to annealing with Co1 El DNA fragments obtained by digestion with the endonuclease HaeIl, which cleaves Co1 El DNA into six fragments (A to F) (Oka & Takanami, 1976). As shown in Table 1, the 7 S DNA hybridized to all classes of the Hue11 fragments, which were immobilized separately on nitrocellulose filter discs. The labelled molecules that hybridized to the Hoe11 fragments were eluted from each filter disc and subjected to reannealing with the separated strands of Co1 El DNA. As shown in Table 2, all species of the 7 S DNA separated by hybridization with the Hue11 fragments contained fragments hybridizing to both the L and H strands, although the proportions were significantly different,. These results show that molecules replicated t’o various ext’ents contained the 7 S DEL4 fragments on both strands. Co1 El DNA replication has been shown to be initiated from the middle region of the Hue11 E segment with the rifampicin-sensitive synthesis of an L DNA fragment of approximately 6 S, which is hybridizable to the E and C fragments (Tomizawa et uE., 1977). The DNA pulse-labelled after 35 minutes of addition of rifampicin contained L DNA fragments hybridizable to the E fragment (Table 2). The L DNA4 fragments hybridized also to the C fragment, but not to the B fragment (data not shown). When rifampicin was added some time aft’er the start- of incubation to the reaction mixture with glycerol and spermidine. a significant amount of early replicative intermediates was formed in the presence of the drug, alt’hough the addition at TABLET Hyhridizution

of the 7 S DNA DNA

used

Co1 El (100%) E fragment (5.4%) C fragment (18.0%) F fragment (1.2%) D fragment (11.0%) H fragment (27.5%) &4 fragment (36.9%)

fragments

with the HaeII

Radioactivity hybridized (cts/min)

fragments

of Co1 El DNA

“6 Hybridized

471 113 235

88 %I 44

74 81 63

14 15 12

DNA pulse-labelled with the Zig-ts extract at 32°C was prepared as described in the legend to Fig. 7, and the DNA in the 7 S fraction of the alkaline sucrose gradient (Fig. 7(d)) was pooled. After neutralization the DNA mixture was used, without further treatment, for annealing with the A to E fragments of Co1 El DNA cleaved by HaeII, which were immobilized separately on filter discs, as described in Materials and Methods. The HaeII fragments shown in the Table are arranged in order of replication. The relative mass of each HneII fragment is presented in parentheses as a percentage of the mass of the unit Co1 E 1 DNA molecule.

3Y-i

T.

SABAKIBAKA ‘FABLE 2

Composition

of the L and H strands in the 7 S Dhl.4 jkagments hybridization tiith the HaelI fragments

Hue11 fragments used for tist hybridization E c D B A

Input

radioactivity (cts/min) 522 834 232 296 344

Radioactivity

(ots/min)

L strand 60 106 43 48 62

separated by

hybridized

to

H strand 181 235 62 67 63

The 7 S DNA fragments isolated from the DNA pulse-labelled with the Zig-t8 extract at 32°C were first subjected to annealing with the Has11 fragments of Co1 El DNA, as described in the legend to Table 1. The labelled DNA hybridized to each Hue11 fragment was eluted from filter discs and subjected to reannealing with the L and H strands of Co1 El DNA, as described in Materials and Methods.

the start of incubation resulted in complete inhibition of the synthesis (data not shown). The rifampicin-sensitive reaction thus was not necessarily followed immediately by initiation of DNA synthesis. The molecules replicating in the lig-ts extract at 32°C contained pulse-labelled strands of various lengths, in addition to the 7 S DNA fragments (Fig. 7(d)). To examine whether the longer DNA chains consisted of both strands, the pulse-labelled DNA, fractionated into five portions according to size in an alkaline sucrose gradient, was subjected to annealing with the separated strands of Co1 El DNA. The results are shown in Figure 8. The labelled DNA in the unit length fraction hybridized to both strands almost equally. The labelled molecules in the fractions of intermediate region between 17 S (unit length) and 7 S also hybridized to both strands. However, these molecules contained more H than L strands. The DNA in the 7 S fraction, on the other hand, contained more L than H strands. Thus the 7 S DNA fragments as well as the longer DNA chains contained both L and H-strand components, but the 7 S DNA fragments contained more L strand while the longer DNA chains were enriched in the H strand. (e) Different

sensitivities

to nicotinamide mononucleotide repair of apurinic sites

of chain elongation

and

To examine whether formation of short DNA fragments in cell extracts was due to incomplete repair of nucleotides misincorporated in the progress of replication, CC molecules of Co1 El DNA containing apurinic sites were incubated in the standard reaction mixture containing an extract from YSl cells and rifampicin. As shown in Table 3, the CC molecules containing apurinic sites, which were susceptible to strand breakage in alkaline solution (Lindahl & Andersson, 1972) were converted to alkalistable CC molecules during incubation for 20 minutes at 30°C. The conversion, however, was completely blocked by the addition of nicotinamide mononucleotide. During incubation in the presence of 10 mM-nicotinamide mononucleotide added instead of NAD, the CC molecules containing apurinic sites were converted to OC molecules, although alkali-stable normal CC molecules remained in the closed circular

DISCONTINUOUS

REPLICATION

Sedrnentation

coefflclent

OF

COL

El

38.5

DNA

(S)

PIG. 8. Hybridization of pulse-labelled DNA fractionated by alkaline sucrose gradient centrifugation with the separated strands of Co1 El DNA. DNA pulse-labelled wit,h the Zig-h extract at, 32°C was prepared as described in the legend to Fig. 7. The DNA was fractionated by alkaline sucrose gradient centrifugation, and the fractions were separately pooled into 6 portions as indicated in the Figure. The DNA of each portion (410 to 2040 ctsjmin) was subjected to annealing with the L and H strands of Co1 El DNA, as described in Materials and Methods. The amounts of the labelled H (0) and L ( ) strand components in each portion, presented on the right-hand side of the Figure, were calculated using the following equation :

cts/min total When hybridized ---*-,

cts/min

hybridized hybridized

to the L or H strand to the L and H strands

b: total

unfractionated DNA (525 cts/min) was subjected to the L and H strands was 116 and 116 cts/min, Radioactivity of each fraction of the gradient..

cts/min

to annealing, respectively.

in each portion. the

amount

of DNA

form during the incubation (Table 3). These results suggest that the cell extract had activities capable of cleaving DNA strands of molecules containing apurinic sites and of repairing the cleaved DNA through a step sensitive to nicotinamide mononucleotide. An endonuclease specific for apurinic sites in double-stranded DNA has been found in E. coli (Verly et al., 1973), and t’he DNA cleaved by the endonuclease has been shown to be repaired by DNA polymerase I and T4 DNA ligase (Verly et al., 1974). Addition of nicotinamide mononucleotide led to conversion of the CC molecules containing apurinic sites to OC molecules. When the reaction mixture containing nicotinamide mononucleotide was supplemented with ATP-dependent T4 DNA ligase, the alkali-labile CC molecules were converted to alkali-stable CC molecules (Table 3). Thus, nicotinamide mononucleotide blocked the joining activity of E. coli DNA ligase for completion of the repair of apurinic sites. Therefore, if nucleotides were misincorporated in the progress of Co1 El DNA replication in a cell extract, followed by repair of the errors, the addition of nicotinamide mononucleotide to the replication system should lead to an excess accumulation of labelled short DNA fragments. The addition of 10 mM-nicotinamide mononucleotide, instead of NAD, to the standard reaction mixture containing an extract from YSl cells, however, led t,o the accumulation of OC molecules containing an interruption at, the origin-terminus

3Hti

T.

SAKAKIBAHA TABLE

Repair of Co1 El DNA

3

condaining apurinic,

sites in a cell extract

C’omposition Cd El DNA used

Incubation with the standard reaction mixtuw

Alkali-st>able, ctlosed circle

None

DNA containing apurinio sites

Normal DNA

+ rifampicin + rifampicin + nicotinamide mononucleotide + rifampicin nucleotide ligase None + rifampicin + rifampioin nucleotide

+ nicotinamide mono+ ATP-dependent T4

+ nicotinamide

mono-

of monomeric

molecules ((7;)

Alkali-labile, closed circle

Open circle

10 97

85 .-c 1

5 3

7

4

89

94

..‘I.

6

89 98

7
4 2

85

.: 1

15

14C-labelled CC Co1 El DNA containing apurinic sites (3700 cts/min and about 0.6 pg) OP3H-labelled normal CC Co1 El DNA (6600 cts/min and about 0.02 rg) was incubated in the standard reaction mixture (300 ~1) containing a YSl cell extract and rifampicin (IO pg/ml) 01‘ with 10 mM.nicotinamide mononucleotide added instead of NAD at 30°C for 20 min. The effect of addition of T4 DNA ligase (0.5 unit) to the reaction mixture containing nicotinamide mononucleotide was also examined. During incubation under these conditions, no significant amount of DNA added was rendered acid-soluble. The structure of DNA extracted after incubation as well as the DNA substrates was analysed by neutral and alkaline sucrose gradient centrifugation. DNA samples for alkaline suwose gradient centrifugation analysis were treated with 0.3 M-NaOH for 10 min at room temperature before sedimentation. All DNA samples with and without incubation were exclusively composed of CC and OC molecules. The proportion of alkali-stable CC molecules in a DNA sample was obtained from the percentage of radioact’ivity in the fraction of collapsed CC molecules in an alkaline gradient. The proportion of alkali-labile CC molecules was calculated by subtraction of the proportion of collapsed CC molecules in an alkaline gradient from that of CC molecules in a neutral gradient, or subtraction of the proportion of OC molecules in a neutral gradient from that of molecules containing single-stranded linear DNA in an alkaline gradient.

region in the newly synthesized L or H strand without significant increase of the fraction of molecules containing labelled short DNA fragments (data not shown), as observed with an extract from YSlO(Co1 El) cells (Sakakibara & Tomizawa, 1974c). When DNA labelled with the Eig-ts extract at 25°C for 60 minutes in the presence of 10 mlcl-nicotinamide mononucleotide added instead of NAD was analysed by alkaline sucrose gradient centrifugation, it separated into approximately 45% single-stranded linear molecules of 17 S (unit length), 10% those of various sizes between 17 S and 6 S, and 45% 6 S fragments (data not shown), similar to the DNA formed in the absence of nicotinamide mononucleotide (Fig. 3(b)). M isincorporation of nucleotides followed by elimination of the nucleotides in the in vitro system must therefore be rare. Taking this result into account, the 7 S DNA fragments found with the Zig-h extract are unlikely to have been formed as a result of incomplete repair of misincorporated nucleotides. The transient accumulation of 7 S L and H DNA fra,gments in the Zig-ts extract thus shows that both strands of Co1 El DNA replicate in a discontinuous manner.

DISCONTINUOUS

REPLICATION

OF

~01,

~1

DNA

387

4. Discussion A cell extract containing thermolabile E. coli DNA ligase ws able to carrJT out iL complete round of GOI El DNA replication at 25°C. However, elongation of the Progeny strands in the lig-ts extract was greatly retarded at this temperature, and depressed at 32°C. Replication of Co1 El DNA thus depends on E. c&i DNA ligase. Taking the action of DNA ligase into consideration (Gellert et al., I968 ; Ohera et al., 1968: Modrich & Lehman, 1973), the retardation as well as depression of chain elongation in the Zig-ts extract seems to be caused by slow joining of nascent short DNA chains. Joining of nascent short DNA chains may be required for further progress of replication. The defect’ in joining of DNA chains would cause some degradation of the molecules replicated in the Zig-ts extract at the higher temperature. The lig-ts extract was much less active in closure of the progeny strands of newly formed OC molecules than the Zig+ revertant extract. Thus E. coli DNA ligase is also involved in the process of the final sealing of progeny strands. This process is one of the rate-limiting processes in the in vitro replication system, and occurs after segregation of daughter OC molecules (Sakakibara & Tomizawa, 1974c; Sakakibara et al., 1976). The final sealing of progeny strands is blocked by nicotinamide mononucleotide. but the compound does not interfere with elongation of progeny strands, and permits the formation of daughter OC molecules (Sakakibara & Tomizawa, 1974c). These results suggest that E. coli DNA ligase is functioning in Co1 El DNA replication with different sensitivities t,o nicotinamide mononucleotide at the stages of chain elongat.ion and t,he final sealing of progeny strands. DNA ligase functioning in the progress of replication seems to become insensitive to the effects of nicotinamide mononucleotide by forming a complex with other replication factors. In the process of conversion of single-stranded phage #Xl74 DNA to double-stranded closed circula’r replicative intermediates in ether-treated cells, it, has been shown that nicotinamide mononucleotide also blocks only the process of conversion of double-stranded open circular replicative intermediates to closed circular ones. although transient accumu1at)ion of short, DNA chains has been observed during the conversion (Hess et al., 1973). E. coli DNA ligase was also involved in the process of the repair of Co1 El DNA containing apurinic sites in cell extracts. The joining activity of the enzyme for completion of the repair was blocked by nicotinamide mononucleotide. The joining mechanism in t,he process of the repair is, therefore. similar to that in the process of the final sealing of progeny strands rather than that in the progress of replication. The difference in the sensitivity t,o nicotinamide mononucleot,ide of chain elongation and repair of apurinic sites implies bhat short, DNA fra,gmcnbs found in the in h-o replication system are not generated in the process of repair of misincorporated nucleotides. Fragmentation of newly synthesized strands containing misincorporated nucleotides has been suggested in E. coti wit,h reduced dl:TPase activity (T.ye et al.. 1977). Replication of Co1 El DNA is initiated with the rifampicin-sensitive synthesis of an L DN’A fragment of approximately 6 S from a lixcd region (Tomizawa, 1975). The fragment is synthesized continuously in a single piece, since early replicative intermediates formed under the condit’ion deficient in DNA ligase conbained the 6 S DNA fragments. Some of the early replicative intermediates contain DNA fragments on both strands of the replicat,ed region (Tomizau-a it al.. 1974). The synthesis of the L DSA fragment is t,hus followed by t,he synthesis of an H DNA fragment in the correspnnding region. The process of replicat*ion following the formation of early

replicative intermediates is a rate-limiting step. and is blocked by the addition of 1974h). Further replication from glycrrol and spermidine (Sakakibara 8~ Tomizawa, early replicative int,ermediates to the completion of replication is insensitive t0 rifampicirl (Sakakibara & Tomizawa. 1974u). Cnder the conditions deficient in DNA ligase, molecules replicated t,o various extents contained short DNA fragments of approximately 7 S on both strands. Since Co1 El DNA replication proceeds unidirectionally (Tomizawa et al., 1974), the above result shows that’ both strands of Co1 El DNA replicate in a discontinuous manner. Replication of Co1 El DNA thus proceeds sequentially with the rifampicin-insensitive sgnt)hesis of DNA fragments of about 500 nucleotide length on both strands. Molecules replicating in the Zig-h extract at 32°C contained pulse-labelled strands of various lengths, in addition to 7 S DNA fragments (Fig. 7). The longer DNA chains contained more H than L-strand components, in contrast to the composition of the 7 S DNA fragments. This difference in the 7 S and longer DNA chains seems unlikely to be caused by some endonuclease attacks on newly formed longer L strands, since the labelled replicating molecules had intact template strands. Even if there were some exonucleolytic digestion of newly synthesized strands, it would not cause the opposite difference in L and H strand components between the 7 S DNA fragments and the longer DNA chains. Thus, the processes of elongation of the L and H strands seem to contain some aspects different from each other. Since the L strand of Co1 El DNA elongates in the direction corresponding t’o the overall direction of unidirectional replication (Tomizawa, 1975), synthesis of an L DNA chain seems unlikely t,o follow the synthesis of an H DNA chain in the corresponding region. Therefore, partly replicated molecules containing a long H DNA chain presumably contained shorter L DNA chains including the 7 8 L DNA fragment(s) in the corresponding region. joining of nascent short DNA chains Such molecules would be formed by preferential for the progeny H strand, or by more frequent synthesis with longer stretches for the progeny H strand. Zn the process of replication of E. c&Y chromosome (Herrmann et al., 1972; Louarn & Bird, 1974) and phage P2 DNA (Kurosawa & Okazaki, 1975), short DNA chains of different size classes for bot,h strands have been found under conditions deficient in DNA polymerase I. In these cases, the strand elongating in the overall 5’-to-3’ direct)ion contains longer DNA chains, in cont,rast to the case of Co1 El DNA replication. The mechanism which distinguishes t,his aspect of the difference in these replication systems is st#ill unknown. The author thanks Dr J. Tomizawa for many valuable suggestions and discussions throughout this work. The author is indebted to Drs M. Gellert and J. Tomizawa for supplymg a Zig-ts strain. Thanks are also due t,o Dr M. Takanami for the HaeII fragments of Co1 El DNA and to Drs T. Inoue and ‘I’. Kada for Col El DNA containing apurinic sites. The author thanks Dr T. Nagata for helpf~~l discussions in the preparation of the manuscript and also Dr M. Gellort for critical reading of ttlc manuscript and llelpf\ll comments. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Xdncation of Japan. 1ZEFERENCES Denhardt, D. T. (1966). Biochem. Biophys. IZes. Common. 23, 641-646. Epstein, W., Jewett, S. & Pox, C. 14’. (1970). J. Bacterial. 104, 793-797. Gellert, M., Little, J. W., Oshinsky, C. K. & Zimmerman, S. B. (1968). Gold Spring Symp. Quant. Biol. 33, 21-26. Gottesman, M. M., Hicks, M. L. & Gellert, M. (1973). J. Mol. Biol. 77, 531-547.

Harbor

DISCONTINUOUS

REPLICATION

OF (‘OL

El

DNA

3X9

Herrmann, R., Huf, J. & Bonhoeffer, F. (1972). iVature ~Vew Viol. 240, 2355237. Hess, U., Diirwald, H. & Hoffman-Berling, H. (1973). J. Mol. Biol. 73, 407-423. Inselburg, J. (1974). Proc. Nat. Acad. Sci., U.S.A. 71 2256 2259. Inselburg, J. 8s Oka, A. (1975). J. Bacterial. 123, 739 1742. Konrad, E. B., Modrich, P. & Lehman, I. R. (1973). J. ;Mol. Hiol. 77, 519-529. Kurosawa, Y. & Okazaki, R. (1975). J. Mol. Biol. 94, 229-241. Lindahl, T. & Andersson, A. (1972). Biochemistry, 11, 361%3623. Louarn, J.-M. & Bird, R. E. (1974). Proc. Nat. Acad. Sci., 0‘S.A. 71, 329-333. Lovett, M. A., Katz, L. & Helinski, D. R. (1974). Nature (London), 251, 337--340. Modrich, I?. & Lehman, I. R. (1973). J. Biol. Chem. 218, 7502 7511. Oka, A. & Takanami, M. (1976). Nature (London), 264, 193-196. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, Ii., Kainuma, R.. Sugino, A. & Iwatsuki, N. (1968). Cold Sping Harbor Symp. Quant. Biol. 33, 12!)-143. Okazaki, It., Sugino, A., Hirose, S., Okazaki, T.. Imae, Y., Kainuma-Kuroda, R., Ogawa, T., Arisawa, M. & Kurosawa, Y. (1973). In DNA Synthesis in vitro (Wells, R. D. & Inman, R. B., eds), pp. 83-104, University Park Press, Baltimore. Olivera, B. M., Hall, Z. W., Anraku, Y., Chien, *J. R. & Lehman. 1. R. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 27-34. Pauling, C. & Hamm, L. (1968). Proc. Nat. Acad. Sci., U.S.A. 64, 1195-1202. Sakakibara, Y. & Tomizawa, J. (1974u). Proc. Nut. Acad. Sk., U.S.A. 71, 802-806. Sakakibara, Y. & Tomizawa, J. (19746). Proc. Nat. L4cad. Sci., U.S.A. 71, 1403-1407. Sakakibara, Y. & Tomizawa, J. (1974c). Proc. Nat. Acad. Sci., U.S.A. 71, 4935-4939. Sakakibara, Y., Suzuki, K. & Tomizawa, J. (1970). .I. Mol. Biol. 108, 569-582. Staudenbauer, W. L. (1974). Nucl. Acids Res. 1, 1153-1164. 257, 253-254. Tomizawa, J. (1975). Nature (London), Tomizawa, J. & Ogawa, T. (1968). Cold Spring Harbor Symp. Q,uant. Biol. 33, 533-551. Tomizawa, J., Sakakibara. Y. & Kakefuda, T. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 2260-2264. Tomizawa, J., Sakakibara, Y. & Kakefuda, T. (1975). I’roc. Nat. AC&. Sci., U.S.-A. 72, 1050-1054. Tomizawa, J., Ohmori, H. & Bird, R. E. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 1865-1869. Tye, B.-K., Nyman, P.-O., Lehman, I. It., Hochhauser, S. & Weiss, B. (1977). Proc. Nut. Acad. Sci., U.S.A. 74, 154-157. Verly, W. G. & Rassart, E. (1975). J. Biol. Chem. 250, 8214-8219. Overly, W. G., Paquette, T. & Thibodeau, L. (1973). Nature New Biol. 244, 67-69. Verly, W. G., Gossard, F. & Crine, P. (1974). hoc. h’at. Acad. Sci., U.S.A. 71, 2273-2275. Yoshimori, R. N. (1971). Ph.D. thesis, University of California, San Francisco.