Multiple promoters for transcription of the Escherichia coli DNA topoisomerase I gene and their regulation by DNA supercoiling

J. Mol. Biol. (1988) 202, 735-742

Multiple

Promoters for Transcription of the Escherichia coli DNA Topoisomerase I Gene and Their Regulation by DNA Supercoiling Yuk-Ching

Tse-Dinh and Rita IL Beran

E. I. du Pont de Nemours and Co. Research and Development Department Experimental Station, Wilmington, DE 19898, U.S.A. Central

(Received 28 July 1987, and in revised form 9 February

1988)

There are four transcriptional promoters present in the 5’ control region of the Escherichia coli DNA topoisomerase I (topA) gene. These were identified with Bal31 nuclease-generated deletions and mapping of the 5’ ends of the mRNAs with avian reverse transcriptase. Recombinant plasmids with all or some of these promoters fused to the galactokinase (gaZK) gene-coding region have been constructed and used to study transcription from the promoters both in vitro and in vivo. The promoter (Pl) closest to the starting ATG codon has a near consensus -35 sequence (GTTGATA) but unusual - 10 (CATATCG) sequence. The other three promoters (P2, P3 and P4) are clustered together 60 base-pairs further upstream. Negative DNA supercoiling is required for efficient transcription from Pl, PI +P2 +P3+P4, P2+P3+P4, P3+P4 and P4 alone. The combination of all four promoters demonstrates greater supercoiling dependence than does any of the other subsets test’ed.

1. Introduction

for the two subunits of DNA gyrase, a type II DNA topoisomerase in E’. coli that can utilize the energy Escherichia coli DNA topoisomerase I, previously from ATP hydrolysis to introduce negative superknown as the o protein (Wang, 1971), has been helical turns into DNA (Gellert et al., 1976, 1977; studied extensively. It catalyzes the interconversion Cozzarelli, 1980; Drlica, 1984). The identifications of different DNA topoisomers by the breaking and of these compensatory mutations have led to the rejoining of a single DNA strand while allowing view that E. coli DNA topoisomerase I, together strand passage to occur. It therefore belongs to the with DNA gyrase, maintain intracellular DNA at a class of DNA topoisomerases designated as type I negative superhelical level that is required for (for a review, see Gellert, 1981; Wang, 1985). The normal cell growth (for a review, seeVosberg, 1985; different topoisomerization reactions it can carry Wang, 1985). out have been described in detail (Wang & Liu, Transcription is one of the important cellular 1979; Tse & Wang, 1980; Brown & Cozzarelli, 1981; processes that is affected by DNA superhelicity Low et al., 1984; Kirkegaard & Wang, 1985). It is (Wang, 1983, 1985). Various promoters respond to encoded by the topA gene, which has been cloned DNA superhelicity changes differently. depending and mapped (Sternglanz et al., 1980; Trucksis & on the promoter sequence, intracellular RNA Depew, 1981; Wang & Becherer, 1983). The polymerase concentration, presence of regulatory nucleotide sequence of the coding sequence and its factors and which of the steps in the polymeraseflanking regions has been determined (Tse-Dinh & promoter interaction is the rate-determining one. Wang, 1986). Transcription from the promoters of E. coli gyrA Even though mutants in which the entire topA and gyrB genes has been shown to increase when gene has been deleted can be isolated (Sternglanz et the DNA template is more relaxed (Menzel & al., 1980), these mutants are only viable in the Gellert, 1983). Since DNA gyrase is required for the presence of compensatory mutations (DiNardo et negative supercoiling of DNA, a homeostatic al., 1982; Pruss et al., 1982; Raji et al., 1985). Some control mechanism was proposed (Menzel & Gellert, of these compensatory mutations have been 1983). In contrast, the transcription of the E. coli mapped to the loci of gyrA and gyrB, which code topoisomerase I gene was shown to be more efficient 735 0022-2836/88/16073+08

$03.00/O

0 1988 Academic

Press Limited

736

Y.-C. Tse-Dinh and R. K. Beran

when the DNA template is more negatively supercoiled (Tse-Dinh, 1985). Since topoisomerase I relaxes negatively supercoiled DNA, this is consistent with a model in which the enzyme activity is regulated by DNA supercoiling and contributes to the overall homeostatic control of DNA superhelicity. When the nucleotide sequence of the topA gene was reported, evidence for the existence of more than one promoter capable of initiation of transcription in the 5’ control region of the gene was presented (Tse-Dinh & Wang, 1986). Here we present data on the mapping of the exact points of transcription initiation as well as in-&o and in-vitro characterization of the multiple promoters we have identified.

2. Experimental

digested with HpaI and HindIII. The ends of the DNA were filled in with E. coli Klenow fragment before electrophoresis in a 1 o/o (w/v) low melting point agarose gel with 50 mM-Tris * HCl (pH ,8), 20 mM-sodium acetate, 1 mM-EDTA. The larger fragment was excised and ligated as described (Tse-Dinh, 1985). pBDR3 was constructed by similar procedures using the restriction enzymes BamHI and HpaI. We also generated derivatives of pBDR2 and pBDR3 with different amountsof nucleotidesequenceproximal to the HpuI site deleted: 10 pg of pBDR1 was first digested with HpaI, then treated with Ba131 nuclease (2.5 units; New England BioLabs) for 10 min. The DNA was extracted with phenol and precipitated with ethanol before the second restriction digest with either Hind111 or BamHI. The DNA was then ligated and used to transform E. coli C600. Colonies were picked from MacConkey indicator plates containing 1 y. (w/v) galactose and their colors were noted. The extents of the deletions were determined by DNA sequencing reactions (Zagursky et al., 1985).

(a) Constructionof the topA-galactokime fusion plaamids The maps of the topA-galactokinase fusion plasmids used in this study are shown in Figure 1. The details for the construction of pBDR1, where a 513 base-pair fragment from the topA gene (denoted as nucleotides 836 to 1348 by Tse-Dinh & Wang, 1986) was placed in front of the galK gene so that production of galactokinase from the plaamid was directed from the 5’ control region of the topA gene, have been reported (Tse-Dinh, 1985). There is a single Hpal site on the plaamid cutting at position 1170. To construct pBDR2, 2.5 pg of pBDR1 DNA was

(b) Bacterial strains

E. coli strain

C600 (gaZK-) was from Pharmacia. Unless otherwise stated, it wa used for the growth of the topA-galactokinase fusion plasmids and meaaurement of galactokinase activities. A C600 AhimA strain was constructed by transduction with Pl lysates prepared from strain K1299 (AhimA82 with a TnlO element from one end of which a deletion extends into the himA gene; Friedman et al., 1984) from Dr S. Bear at du Pont. Following selection for tetracycline-resistant (TnlO-

ori

I. HpaI/BamHI

I

HmdllI

2. 3.

Klenow T4 ligose

/h’paI

t

OrI

ori

Figure

1. Plasmids

topA gene is indicated

with the control region of topA fused to the galactokinase by a bold line.

(galK) coding region. Sequence from the

Multiple

Promoters for E. coli topA Gene

containing) recombinents, all were confirmed to have the himA mutation by their inability to support growth of mu phage. GP200 (gyrA (Nal’) gyrB225 (topAcyd3) 204) was obtained from Dr K. Drlica of the Public Health Research Institute of the City of New York. (c) Mapping

of stmt &3!? of tfumcription

RNA wa prepared from E. coli C600 cells containing either pBR322 or pBDR1 grown in LB medium (Miller, 1972) at 37°C. Cells were harvested when the A,,, value was around 1 (Salser et al., 1967). An oligonucleotide complementary to nucleotides 1327 to 1344 of the topA gene (Tse-Dinh & Wang, 1986) was synthesized by the solid phase method and labeled at the 5’ end with phage T4 polynucleotide kinase and [Y-~~P]ATP. Then 100 pg of the RNA w&8 mixed with 500 ng of the primer and precipitated with ethanol. The pellet was resuspended in 20 ~1 of 60 mr+r-Tris.HCl (pH 83), 75 mM-NaCl, 7.5 mMMgCl,, 0.5 mnn-dithiothreitol and 1 rnM of each of the 4 deoxynucleotide triphosphates. Then 25 units of AMV reverse transcriptase were added. The mixture was incubated at 42°C for 10 min and 20 ~1 of 80% (v/v) formamide, 10 mM-NaGH, 0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyan01 was added. The mixture was heated at 100°C for 2 min before analysis by electrophoresie. Sequencing reactions using the same primer with pBDR1 aa substrate were loaded in adjoining lanes of the same gel (Zagursky et aE., 1985). (d) Guhctokinuse

uasuys and in-vitro

trunscriptim-

translation reactions with [35S]methionine These were performed as described (Tee-Dir& 1985), except galactokinase units measuredin vivo when the cells were treated with oxolinic acid was normalized per mg of soluble protein in lysate instead of per optical density unit of cells. Protein concentration was determined by the BioRad protein assay system. analyzed an LKB Autoradiographs were with densitometer.

3. Results (a) The topA gene has multiple promoters In trying to localize the promoter for the topA gene, we found that different portions of the DNA upstream from the coding sequence can direct transcription initiation and allow expression of galactokinase (Tse-Dinh & Wang, 1986). The presence of multiple promoters was confirmed by primer extension. on in-vivo-synthesized RNA with reverse transcriptase. Four bands were observed when the RNA was from cells containing pBDR1 and not seen with RNA from cells containing pBR322 (Fig. 2(a)). We do not know if the relative intensities of these bands reflect accurately the utilization relative of the four promoters. Extensions from mRNA from the chromosomal copy of the topA gene were not detectable with the experimental procedures employed, and were probably due to insufficient sensitivity. The putative transcriptional start sites that the four bands correspond to are shown in Figure 2(b). The most plausible promoter sequence for each transcriptional start site was searched for with a

737

promoter search algorithm (Mulligan et al., 1984) and is shown in Figure 2(b). (b) Deletion analysis of the topA control region pBDR2 contains sequence to the right of the HpaI site while pBDR3 contains sequence to the left. Derivatives of these plasmids with Hal31 nuclease-generated deletions were constructed. The extents of these deletions and the phenotype of C600 cells containing these plasmids are shown in Figure 3. The galactokinase expressions observed are in agreement, with the positions of the promoters assigned with the transcriptional start site results. Deletions past the -35 region on Pl and P4 abolished expression in vivo as the colonies these plasmids were white on containing MacConkey plates containing galactose. (c) Dependenceof transcription on DNA supercoiling We have chosen to further quantitate the expressions from the promoters on the different plasmids constructed by in-vitro transcriptiontranslation using [3sS]methionine. If relaxed DNA is used as a starting substrate, and the gyrate activity in the 530 extract is inhibited by Novobiocin, the DNA remains fully relaxed during the synthesis of labeled galactokinase, allowing comparison between supercoiled and relaxed substrates. Our previous data have shown that synthesis of /.?-lactamase from the amp gene on these plasmids, when measured with this in-vitro transcription-translation system, were independent of DNA supercoiling (Tse-Dinh, 1985). Therefore, we compared the expression from the topA promoters with the ratio of galactokinase to fl-lactamase synthesized in the same reactions. This was measured by densitometer scanning of the autoradiographs of the SDS/ olyacrylamide gel after electrophoresis of the 3PS-labeled proteins. The results are shown in Table 1. In general the results were reproducible to within lOo/o, except in the cases where the galactokinase/fl-lactamase ratios were less than 0.1. There the results were reproducible to within 35% of the values indicated. Expression from supercoiled substrates was highest in pBDRI where all four promoters are present. With relaxed DNA, expression from pBDR1 was as low as any of the other plasmids. It therefore had the greatest change in amount of transcription with change in DNA superhelicity. We have also measured galactokinase expression in vivo with C600 cells containing pBDR1, pBDR2 and pBDR3. The results are shown in Table 2. Qualitatively, expression from all three plasmids decreased when gyrase activity was inhibited by treatment with oxolinic acid, but the decrease in pBDR1 expression, in particular, was not as dramatic as that observed in vitro. This is probably because oxolinic acid inhibits both the supercoiling and relaxing activities of gyrase so only a small change in DNA supercoiling resulted. We have

738

Y.-C. Tse-Dinh and R. K. Beran

CTA

G

I

2

(0) 1001

CGCTGGTGGC

1051

( -35 AAAGTTGCGT

1101

TGGCAA’i’AGA

T+TGCTT*&?%CGACCAA

1151

CC?GTAGGCC

AAGACCTGTT

AAGAGCGCCT

TACTGGCAAC

TTTGGATTTT

P4

. ATCGGATTTT

GCATGCTAAT

-as *

-10

P3

ATCAGGTmGmCT

TTCGTCAATC

P2

-10

-10

AATTCCGTCG

Hpa

T-AGCG

1

1 AACTCAGTCA

CCTGAATTTT

Pl

CGTGAACAGA -10

1201

GTCACGACAA

GGGGT-

TCCGCAGAGA

GCGAGTCC-GGTAAC?

1251

CGTTGCCAGT

GGAAGGTTTA

TCAACGTGCG

ACGCATTCCT

GGAAGAATCA

(b)

Figure 2. (a) loaded onto a autoradiographed. The sizes of the region of topA. transcriptional

Primer extension of RNA with reverse transcriptase. Sequencing and primer extension reactions were 10% sequencing gel and run until the xylene cyan01 was near the bottom of the gel. It was then Lane 1, RNA from C600 cells containing pBDR1; lane 2, RNA from C600 cells containing pBR322. extended molecules in lane 1 are 94, 191, 226 and 256 bases long. (b) Nucleotide sequence of the control The numbering is the same aa reported (Tse-Dinh & Wang, 1986). The arrows indicate the position of start sitesmapped by reverse transcriptase. The brackets indicate the - 10 and -35 regions.

Multiple

Promoters

739

E. coli topA Gene

for

PlasmId

gal K phenotype

P4

836

P3

P2

Pl

1348 f

pBDR1 1171

Pl

1348

1214

Pl

1348

+

pBDR2

t

pBDR2-1 1224

1348

pBDR2-2 836

P4

P3

P2

1170 t

pBDR3 836

P4

P3

1112

pBDR3-1

+ 836

P4

1083 t

pBDR3-2 836

1007

pBDR3-3

Figure 3. Region of the topA sequence present in the galactokinase fusion plaemids. The numbers (as given by TseDinh $ Wang, 1986) indicate the positions of the first and last nucleotides of the sequence. For the galactokinaae phenotype, + indicates the appearance of red colonies when the plasmids were transformed into C600 and plated on MacConkey agar plates containing galactose;- indicates that white colonieswere obtained. extracted plasmids from cells from this experiment using two different plasmid preparation procedures (Twigg & Sherratt, 1980; Holmes & Quigley, 1981) and found that the oxolinic acid treatment did not reduce plasmid copy number. The change in linking number of the plasmid DNA after oxolinic acid treatment was about +4 (data not shown) when examined by agarose gel electrophoresis in the presence of the intercalator chloroquine (Waley, 1974).

In the E. coli strain GP200, DNA is normally less supercoiled than in wild-type. However, DNA supercoiling increases upon treatment with oxolinic acid due to a mutation in the gyrA gene that conferred partial resistance to the drug, as well as mutations in gyrB and topA genes (Manes et al., 1983; Pruss et al., 1986). Plasmids pBDR1, pBDR2 and pBDR3 were transformed into this strain and their galactokinase activities were found to increase upon treatment with oxolinic acid (Table 2). This is

Table 1 Effect

of

DNA substrate he&city on in-vitro expression from

topA-galactokinase

Galactokinase/j-lactamase

Plasmid pBDR1 pBDR2 pUDR2pBDR2-2 pBDR3 pSDR3-1 pRDR3-2 pSDR3-3

Pl, 1

Promoters

Supercoiled DNA?

P2, P3, P4 Pl PI None P2, P3, P4 P3, P4 P4 None

1.77 kO.03 0.51 f 0.02 0.48_+0~05 0 1~00~0~05 0.95 f 0.02 0.55 * 0.03 0@9*0.02

Relaxed DNA + supercoiled$ 036 f 0.09 0.45kO.04 0.39 f o-01 0 0.98 * 0.07 0~70*0~01 0.48+0.03 0.08 f 0.02

fusion

plasmids

ratio DNA + Novobiocin§

Relaxed

0.07 * 0.02 0.11 kO.01 o-09 * 0.03 0 0.27kO.02 0.14+0.02 o~ll*o~ol 0

t The DNA was supercoiled and remained so during the transcription-translation reaction. 3 Relaxed DNA was used and it was converted to negatively supercoiled DNA by the gyrate activity in the cell-free extract during the transcription-translation reaction. 8 The DNA remained relaxed due to the presence of the gyraae inhibitor Novobiocin (0.1 mg/ml). Autoradiographs of different exposures were obtained for densitometer scanning to ensure linear response. Densitometry scans were analyzed by an LKB GelScan XL Laser Densitometer Program. The ratio of the areaa of the integrated peaks corresponding to galactokinase and p-lactamase are shown here. The data represent the average of at least 2 independent experiments and the range is indicated.

740

Y.-C. Tse-Dinh

and R. K. Beran

Table 2

Table 3

Effect of oxolinic acid treatment on galactokinase expression from topA-galactokinase fusions

Eflect of the himAg2 mutation on galactokinase expression from topA-galactokinaae fu&n plasmids

Galactokinaee units (per mg protein)

Strain

Plasmid

Control

c600 C600 c600 GP200 GP200 GP200 GP200

pBDR1 pBDR2 pBDR3 pBDR1 pBDR2 pBDR3

4&Z&38 158k 14 385f21 329f33 183&5 311+19 22*11

Oxolinic acid-treated 262 f 15 8Of8 197510 48Of40 216+4 427 + 23 11+3

Cells were grown as described (Tae-Dinh, 1985) at 37°C and treated with 2 pg oxolinic acid/ml when the A,,, was about 0.4, After another hour of growth at 37°C the cells were lysed and assayed for galactokinase activity (McKenney et al., 1982). 20 ~1 of lysate were added to the reaction mixture. Incubation was at 30°C for 20 min. Treatment with drug and galactokinaee assays were carried out in duplicate for each experiment. The average from at least 2 independent experiments is given here and the range is indicated. Galactokinase units are expressed aa nanomol galactose phoaphorylated/min per mg of protein in lysate instead of per optical density unit of cells because of possible effects of oxolinic acid treatment on the shapes of cells.

in agreement with the model of homeostatic control of topoisomerase I activity and DNA supercoiling in E. coli. We have not attempted to transduce the galKgenotype into GP200. This strain grows poorly due to triple mutations in topA, gyrA and gyrB genes. For instance, we could not obtain any transformants in GP200 with a plasmid that has galK under the control of tat or trp promoters. We have measured the galactokinase activities in GP200 itself and found it to be relatively low and unchanged upon treatment with oxolinic acid (Table 2). The response of the topA promoters to oxolinic acid in GP200 not only confirmed that they were responding to the effect of the drug on DNA that they could supercoiling but also showed respond in the way required for homeostatic control if DNA became more negatively supercoiled than in normal E. coli cells. (d) EfSect of himA mutation Upon examination of the nucleotide sequence of the control region, a sequence (GACAAGGGGTTGATA) that closely resembles the consensus sequence for the integration host factor (1HF)t binding site ((T/C)N(T/C)AANNNNTTGAT(A/T)) (Gardner & Nash, 1986) was found around the - 35 region of Pl between nucleotides 1206 and 1220. To determine if IHF plays a role in regulation of topA expression, several of the topA-galactokinase fusion plasmids were transformed into a C606 AhimA82 strain and galactokinase activities were measured and are

7 Abbreviation

used:

IHF,

integration

host

factor.

Galactokinaae units (per absorbance unit of cells) Plasmid pBDR2 pBDR2-1 pRDR1

C600 4Ok2 32+5 100+7

C600 himA 82k8 66*10 128+11

Cells were grown a8 described (Tse-Dinh, 1985) at 37°C until reached 1. Assays were done a8 described for Table 2. the ho The growth of cultures and galactokinaae assays were carried out in duplicate for each experiment. The average of at least 2 independent experiments is shown here and the range is indicated. Galactokinaae units are expressed as nanomol galactose phosphorylated/min per ml of cells at A,,, = 1.0.

shown in Table 3. Expression from Pl in pBDR2 increased about twofold in the presence of the AhimA82 mutation. The deletion in pBDR2-1 extended into the putative IHF binding site. However, closer inspection of the sequence showed that ligation to the filled-in Hind111 site upstream has recreated a comparable IHF binding sequence. Galactokinase expression from pBDR1 had a 25% increase in the presence of the himA mutation. 4. Discussion The results presented here demonstrate that there are multiple promoters for the transcription initiation of the E. coli DNA topoisomerase I gene. Mapping of transcriptional start sites using in-vivosynthesized RNA showed that four promoters were utilized under the growth condition employed. The presence of these four promoters was consistent with the results from deletions generated in the control region. In-vitro transcription-translation showed that the subset of promoters tested were more efficient when the DNA substrates were negatively supercoiled. The greatest difference between expressions from negatively supercoiled and relaxed DNA was seen with all four promoters present. We do not have P2 or P3 alone on a plasmid so it is possible that by themselves they may not be stimulated by DNA supercoiling. As shown by analysis of E. coli promoter sequences (Hawley & McClure, 1983; Harley & Reynolds, 1987), the most conserved nucleotides in the - 10 region of the consensus E. coli promoter sequence (TATAAT) are the first T, second A and the sixth T nucleotide. Pl lacks both the first T and sixth T nucleotide. Its score (42) in the promoter search algorithm (Mulligan et al., 1984) is therefore the lowest among the four promoters described here. Nevertheless, it represents the only potential promoter sequence of significant score for the that transcriptional start site mapped. The presence of the -35 sequence (TTGATA), with good homology to the E. wZi consensus (TTGACA), was confirmed by the Bal31

deletion

results.

In the most

recent

Multiple

compilation of E. coli promoters (Harley & Reynolds, 1985), the only promoters that lack both the first and sixth T nucleotide in the - 10 region are the heat shock promoters and the promoter for the dapD gene (GATAAA) which is under the regulation of lysine limitation (Richaud et al., 1984). We do not know at present if regulation by supercoiling is the only reason behind utilization of this combination of promoters. It is possible that one or more of the promoters can be repressed or stimulated by physiological conditions that do not affect supercoiling directly. Previous work (Wang & Becherer, 1983) has shown that the truncated topA gene on a multicopy plasmid did not affect expression of the chromosomal copy of the gene significantly. Thus, if trans-acting factors are interacting with the upstream sequences, they are likely to be present in excess under the growth conditions employed there. Yamamoto & Droffner (1985) isolated strict anaerobic and aerobic mutants in Salmonella typhimurium. The strict anaerobic mutants contained a defective DNA topoisomerase I gene, while the strict aerobic mutants contained a defective DNA gyrase subunit A gene. They detected topoisomerase T activity in cell-free extracts of wild-type cells cultured under vigorous aerobic condition, but not in extracts from anaerobically cultured wild-type cells. We find the idea that gene expression may be altered by a different level of topoisomerase I activity under different growth conditions attractive. We hope to understand the problem further by carrying out more detailed studies in vivo. Genetic studies have suggested that IHF can regulate expression of certain genes at the level of translation (Hoyt et al., 1982). Besides its requirement for site-specific recombination, its role in E. coli functions is not completely known. Due to the presence of a sequence near Pl that resembles the IHF binding site, we measured expression of galactokinase from several of our plasmid constructions in strains with and without the himA mutation. Previous work (Gellert et al., 1983) showed that the AhimA82 mutation decreased gyrA expression about fourfold. Double mutations in himA and a certain class of gyrB-him(Ts) decreased DNA supercoiling greatly (Friedman et al., 1984). Our results showed that expression of Pl was increased about twofold with the AhimA82 deletion present. A. 25 yO increase in expression from all four promoters in pBDR1 was observed. This may partly account for the synergistic effect of AhimA82 and gyrB-him(Ts) on DNA supercoiling. Besides the putative IHF binding site, another sequence

between

Pl

and

the cluster

of promoters

P2, P3, P4 that may be a potential recognition site for a regulatory factor, is a sequence of dyad symmetry that extends from nucleotide 1190 to 1207

(TCGTGAACAGAGTCACGA).

We

741

Promoters for E. coli topA Gene

hope

to

examine effects of mutations in the topA control region on topoisomerase I expression and DNA supercoiling.

We thank Drs J. C. Wang, R. Menzel, R. Zagursky and R. LaRossa for helpful comments and discussions, Hanna and Helen Tabb for technical assistance.

Diane

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Raji, A., Zabel, D. J., Laufer, C. S. & Depew, R. E. (1985).J. Bacterial. 162, 1173-1179. Richaud, C., Richaud, F., Martin, C., Haziza, C. & Patte, J.-C. (1984). J. Biol. Chem. 259, 14824-14828. Salser, W., Gesteland, R. F. & Bolle, A. (1967). Nature (London), 215,588-591. Sternglanz, R., DiNardo, S., Wang, J. C., Nishimura, Y. & Hirota, Y. (1989). In Mechanistic Studies of DNA Replication and Genetic Recombination (Alberts, B. M. & Fox, C. F., eds), pp. 833-837, Academic Press, New York. Trucksis, M. & Depcw, R. E. (1981). Proc. Nat. Acud. Sci., U.S.A. 78, 2164-2168.

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by M. Gottesman