THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 5, Issue of February 15, pp. 2304-2309 1987 Printed in ~ . . s . A .

Formation of Rolling-circle Molecules during 4x174 Complementary Strand DNA Replication*

(Received for publication, June 18, 1986)

Minsen MokS and Kenneth J. Mariansg From the Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

The primosome is a mobile multiprotein priming ap- paratus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n‘); and proteins i, n, and n”). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in un- winding duplex DNA strands was investigated in the coliphage 4x174 SS(c) + replicative form DNA repli- cation reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase I11 holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and a-3), which lack primosome assem- bly sites, failed to support significant linear multimer production and that replication of 4x174 with the general priming system (the DNA B and DNA G pro- teins and DNA polymerase I11 holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.

~ ~~~

The replication of duplex DNA molecules requires that the parental strands be unwound at the replication fork. Enzy- matic activities, termed helicases, that denature duplex DNA in an ATP-dependent fashion have been identified in Esche- richia coli and in phage-infected E. coli.

E. coli helicase I (1) is encoded by the F factor (2) and is assumed to be involved in transfer of DNA strands during conjugation. Helicase I1 (3) is the uurD gene product (4-7) and is probably involved in DNA repair. The function of helicase I11 (8) remains unclear. Another E. coli helicase, the product of the rep gene (9), is required for the RF’ + RF and RF + SS(c) stages of 4x174 DNA replication (10, 11) and is

* These studies were supported by National Institutes of Health Grants GM34557 and GM34558. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Robert Wood Johnson Jr. Charitable Trust Fellow in the Center’s Medical Scientist Training Program.

Recipient of an American Cancer Society Faculty Research Award and an Irma T. Hirschl, Monique Weill-Caulier Career Sci- entist Award.

The abbreviations used are: RF, replicative-form DNA; SSB, E. coli single-stranded DNA binding protein; pol 111, DNA polymerase I11 holoenzyme; SS(c), single-stranded circular DNA form 11, relaxed, circular, duplex DNA; form 111, full-length, linear, duplex DNA; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

also possibly involved in DNA repair (12). On the other hand, the phage T7 gene 4 protein (13, 14) and phage T4 gene 411 61 proteins (15-18) are unique enzymes possessing both hel- icase activity (19, 20) and primase activity (15, 21-23). These proteins act at the replication fork while bound to the lagging- strand template functioning both to unwind the parental DNA and synthesize primers that are used to initiate synthesis of Okazaki fragments. The equivalent E. coli replication pro- tein(s) is the primosome (24), a multi-enzyme mobile priming apparatus discovered because of its requirement for the initi- ation of 6x174 SS(c) + RF DNA replication (25, 26). Al- though the DNA G protein-catalyzed priming activity of the primosome has long been documented (25, 26), its function as a helicase has remained equivocal. Recently, LeBowitz and McMacken (27) have demonstrated that the DNA B protein, one of the components of the primosome, has an intrinsic helicase activity.

Studies from this laboratory have demonstrated that low concentrations of topoisomerase I are required to generate template specificity in the reconstituted pBR322 DNA repli- cation system (28). One of the major synthetic products under these conditions was multigenome-length, linear, duplex DNA molecules presumably formed by a rolling circle-type replica- tion mechanism. The appearance of these large molecules required the primosomal proteins.’ Since rolling-circle DNA replication requires a helicase-type activity, it was inferred that the primosome, or some sub-assembly or component of the primosome, was the agent responsible (28).

The 4x174 SS(c) + RF DNA replication system was chosen for preliminary evaluation of the ability of the pri- mosome to act as a helicase because it had simpler protein requirements than the pBR322 DNA replication system and did not generate a complex spectrum of synthetic products. I t was expected that helicase activity would be manifested dif- ferently in the 4x174 DNA replication system because of the use of single-stranded DNA templates as opposed to double- stranded templates: this activity would permit DNA synthesis to progress beyond the form I1 structure by a strand-displace- ment mechanism to create rolling-circle forms. I t should be noted that the structures and generation of replicating rolling- circle DNA molecules considered in the present study are not related to those generated in the RF + SS(c) stage of 4x174 replication that requires the viral gene A protein and the E. coli rep protein (10, 11). In this report, the generation of replicating rolling-circle DNA molecules in the 4x174 SS(c) + RF DNA replication system is demonstrated and evidence is provided that their formation is primosome dependent.

MATERIALS AND METHODS

DNA and Enzymes-4x174 (+) SS(c) DNA was prepared by an established procedure (29). Viral SS(c) DNA of flR208 (30), an fl

J. Minden and K. Marians, unpublished data.

2304

DNA Iielicase Activity A s s ~ i ~ t e d with the E. coli P ~ i r n ~ s o ~ e 2305

recombinant phage (a gift of Dr. P. Model, Rockefeller University), was prepared as previously described (31). The PstI restriction endonuclease was from New England BioLabs. Bovine pancreatic deoxyribonuclease I was from Sigma. O l i ~ n u c l ~ t i d e s were prepared using an Applied Biosystems 380A DNA synthesizer. PstI (5'- AACTCTGCAGGTTG-3 ' ) and PstIII (5"AGCGATAAA- ACTCTGCAGGTTGGATACGCC-3') represented (-1 strand 6x174 DNA sequences at the PstI endonuclease cleavage site. PstII (5'- CAACCTGCAGAG~-3') represented (+) strand sequences at the PstI site.

Replication Proteins-E. coli replication proteins were purified as previously described (28); the specific activities in units/mg are indicated in parentheses: the E. coli single-stranded DNA binding protein (SSB) (485), the DNA B protein ( 7 , 0 ~ ) , the DNA C protein (12,800), the DNA G protein (19,200), the DNA N protein (13,500), DNA polymerase III* (29,700), protein i (2,390), protein n (8,900), protein n" (lO,OOO), and factor Y (114,000). &X174 SS(c) -+ RF DNA R e p ~ ~ a t w ~ - ~ p l i c a t i o n reaction mix-

tures (25 pl ) containing 50 mM Hepes-KOH buffer (pH 8.0 a t 20 "C), 10 mM magnesium acetate, 10 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 2 mM ATP, 100 p~ each of CTP, GTP, and UTP, 40 p~ each of dATP, dCTP, dGTP, and dTTP, 225 pmol (as nucleotide) of &X174 SS(c) DNA, 0.75 fig of SSB, 0.55 unit of the DNA B protein, 0.9 unit of the DNA C protein, 0.6 unit of the DNA G protein, 0.7 unit of protein i, 0.45 unit of protein n, 0.75 unit of protein n", 0.55 unit of factor Y, 1.8 units of DNA polymerase III*, and 0.5 unit of the DNA N protein were incubated at 30 "C. Replication proteins were added last as a combined protein mix to reaction mixtures containing #X174 SS(c) DNA and SSB. Depending on the type of subsequent analysis, reaction products were labeled with either [3HJ dTTP (200-250 cpm/pmol) or [cx-~'P]~CTP (500-5000 cpm/pmol) purchased from Amersham Corp. Reactions containing 13H]dTTP were stopped by the addition of 0.1 ml of 0.2 M sodium pyrophosphate, 0.1 ml of 1 mg/mf heat-denatured salmon sperm DNA (as carrier), and 4 ml of 5% trichloroacetic acid. After 10 min on ice, acid-insoluble material was collected on glass fibers filters (Enzo), which were then washed with 1% trichloroacetic acid and 95% ethanol and dried under a heat lamp. The radioactivity retained was then determined. Reac- tions containing [cv~'P)~CTP were stopped by heating a t 65 "C for 5 min followed by the addition (5 p1/25 pl reaction mixture) of a dye mixture containing 1 mg/ml xylene cylanol, 1 mg/ml bromphenol blue, 20 mM EDTA, 2% (w/v) sarkosyl, and 50% glycerol.

Gel ~ ~ c t r o p ~ r e ~ ~ Andysis of Reaction P ~ ~ t s - L a ~ l e d syn- thetic DNA products were typically resolved by electrophoresis through 0.8% agarose (SeaKem ME) gels at 1.8-5.5 V/cm at room temperature in TAE buffer (50 mM Tris-HC1 (pH 7.9 at 20 "C), 40 mM sodium acetate, and 1 mM EDTA). Gels were then dried under vacuum on a gel-drying apparatus and autoradiographed with Kodak XAR-5 fi1m. Two-dimensional gel electrophoretic analyses were per- formed on a horizontal gel apparatus with 0.8% agarose gels. After electrophoresis in the first dimension under native conditions (2.7 V/ cm for 9 h) using TAE buffer, gels were soaked in four changes of 8 gel-volumes each of 50 mM NaOH, 2 mM EDTA over 1 h at room temperature. Electrophoresis in the second dimension (2.6 V/cm for 8.5 h) was performed in the same apparatus after repositioning the reequilibrated gels by a 90 angle. The running buffer under dena- turing conditions was 30 mM NaOH, 2 mM EDTA. Southern blot analysis (32) of replication reaction products resolved by a two- dimensional gel electrophoresis was performed by transfer with 20 X ssc (1 X ssc = 0.15 M NaCI, 15 mM sodium citrate) to a 0.45 pm nitrocellulose membrane (Millipore) after acid depurination, alkali denaturation, and neutralization. The baked membrane was prehy- bridized for 2 h a t room temperature in 50 mM sodium phosphate buffer (pH 7.0 at 20 "C), 6 X SSC, 0.25% SDS, 2.5 X Denhardt's solution (1 x Denhardt's solution = 0.2 mg/ml bovine serum albumin, 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml Ficoll) and 50 pg/ml denatured salmon sperm DNA. Hybridizations were carried out in the same solution containing 32P-5'-end-labeled single-stranded oli- gonucleotide probes instead of salmon sperm DNA. Autoradiography followed washing for 30 min at 4 "C with 6 X SSC. Oligonucleotides were phosphorylated with [y-32P]ATP (Amersham Corp.) using T4 polynucleotide kinase (Pharmacia P-L Biochemicals) and isolated by gel filtration of a 50-pl sampfe through a 6 X 25-cm Sephadex G-50 column.

RESULTS

Structural Characterization of Linear Multimer Products- The use of the 4x174 SS(c) "+ RF DNA replication reaction to evaluate the ability of the primosome to act as a helicase was based on several observations. The predicted manifesta- tion of a helicase activity present in such a reaction was the generation of rolling circle-type molecules. ~ o m p l e m e n ~ ~ (-) strand DNA synthesis on the input viral (+) strand SS(c) templates could continue beyond the form I1 structures for multiple cycles if a strand-displacement activity were pro- vided. The structure of these products would be relaxed, double-stranded, unit-length circles possessing multigenome- length, linear tails. In fact, Shlomai et al. (33), in a previous study of the 4x174 SS(c) -4 RF DNA replication reaction with purified proteins, described a subpopulation of synthetic products that appeared by electron microscopic analysis to be duplex, unit-length, relaxed circles with duplex, linear tails of various lengths.

The kinetics of [3H]dTMP incorporation into acid-insolu- ble material during 4x174 SS(c) "+ RF DNA synthesis was monitored (Fig. 1). Within 5 min, synthesis of DNA products reached an amount equivalent to that of the input template; however, the reaction continued linearly for another 40 min. After 90 min, the accumulation of synthetic product was 7- fold greater than the amount of input template, suggesting that the reaction products were not solely form I1 molecules.

Two major species of labeled products were identified by native agarose gel electrophoretic analysis (Fig. 2). Form I1 molecules appeared early during the incubation but did not increase in amount significantly thereafter. The second spe- cies of products migrated very slowly and did not begin to accumulate to an appreciable amount until after 10 min of incubation.

In order to obtain an initial appraisal of the structure of the larger molecules, a complete PstI restriction endonuclease digestion of total reaction products was performed. The 4x174 genome possesses a unique PstI cleavage site. Both species of DNA products were converted to form I11 molecules by this treatment, suggesting a repetitive, duplex nature in the larger products (data not shown). This could be further demonstrated by performing a partial PstI endonuclease digestion of the synthetic products (Fig. 3A) . Based on their

400 g

I 4 I

5 0 ~ 15 x) 45 60 75 90

Time (min) FIG. 1. Kinetics of dTMP incorporation into acid-insoluble

material in the 4x174 SS(ce) - RF DNA replication reaction. A 200-p1 (8 X) standard reaction mixture containing 13H]dTTP was incubated at 30 "C. At each time point, a portion of the reaction was stopped as described under "Materials and Methods" to determine the amount of dTMP incorporation per 25 pl. The synthesis of products equivalent to the amount of input DNA corresponded to 225 pmol of nucleotide or 56.25 pmol of dTMP incorporation/25 pl of reaction mixture.

2306 DNA Helicase Activity Associated with the E. coli Primosome

1 2 3 4 5 6 7 . .

.- - u - lineor multimer

A u cl.3) w w r-r <;;;;

FIG. 2. Kinetics of linear multimer formation in the 6x174 SS(c) 4 RF reaction. A 50-pl (2 X) standard reaction mixture containing [ ( u - ~ P I ~ C T P was incubated at 30 "C. At each time point, 6 pl was removed and that portion of the reaction stopped as described under "Materials and Methods." 3ZP-labeled synthetic DNA products were separated by agarose gel electrophoresis at 2.3 V/cm for 10 h. Lane I, 2.5 min; lane 2, 5 min; lane 3, 10 min; lane 4, 20 min; lane 5, 30 min; lane 6,45 min; lane 7,60 min.

A. 1 2 3

0 . 1 2 3 4 5

1 -- 'a I -:Xner -linear

kbp multimer

23 -

9.4-

6.6 -

4.4- n m <;;;; ::I -form I1

FIG. 3. Partial Pet1 restriction endonuclease and deoxyri- bonuclease I digestions of reaction products. A, a standard reaction mixture containing [w3'P]dCTP was incubated at 30 "C for 40 min and stopped by heating at 65 "C for 5 min. "P-labeled products were partially digested with the PstI restriction endonuclease (1 unit per 6 pl) on ice after increasing the concentration of NaCl in the reaction mixture to 100 mM. The digestion products were separated by electrophoresis through a horizontal 0.3% agarose gel in TAE buffer at 1.7 V/cm for 17 h. Lane I, undigested reaction products; lane 2, products digested for 60 s; lane 3, products digested for 120 s. R, a standard reaction mixture was incubated at 30 "C for 40 min and stopped by heating at 65°C for 5 min. "'P-labeled products were partially digested with deoxyribonuclease I on ice for 5 min after the addition of CaCI2 to a final concentration of 0.15 mM to the reaction mixture. The digestion products were separated by electrophoresis through a horizontal 0.3% agarose gel in TAE buffer a t 2.1 V/cm for 15 h. Lane I, undigested reaction products; lane 2, products partially digested with PstI restriction endonuclease; lane 3, products digested with deoxyribonuclease I (1 ng/6 pl); lane 4, products digested with deoxyribonuclease I (3 ng/6 pl); lane 5, products digested with deox- yribonuclease I (10 ng/6 pl).

relative electrophoretic mobilities, the molecular weights of the molecules generated by this treatment appeared to differ by integral multiples of the unit 4x174 genome size. At least 15 discretely resolved species could be discerned.

Two types of structures could conceivably account for the pattern generated by partial digestion with the PstI endonu- clease: a long, linear molecule containing tandemly repeated 4x174 genomic sequences or a catenated array of unit-length 4x174 circles. If the latter possibility were the case, limited digestion by pancreatic deoxyribonuclease I should result in a discrete pattern identical to that generated by partial diges- tion with the PstI endonuclease. Agarose gel electrophoresis of synthetic products treated with low concentrations of DNase I resulted in a smear rather than a pattern containing discrete species, eliminating the possibility that the large DNA product was a catenated structure (Fig. 3 B ) . Based on

the results of the partial digestion by the PstI endonuclease, the size of the large DNA was estimated a t 100 kilobases. Multigenome-length, linear products of this type were an indication of a rolling-circle mechanism occurring during the replication reaction.

The conversion of these linear multimers to form I11 prod- ucts by complete digestion with the PstI endonuclease, which does not cleave single-stranded 4x174 DNA (34), implied that they were double-stranded in structure. In addition, these large molecules were sensitive to digestion by exonuclease I11 but not by nuclease S1 (data not shown).

Additional structural information was gathered from anal- ysis of two-dimensional agarose gel electrophoretic separation of the reaction products. This method was chosen to allow examination of the larger products separate from the form I1 molecules. First dimensional electrophoreses were under na- tive conditions, while second dimensional electrophoreses were under alkaline denaturing conditions. The large rolling- circle molecules could be resolved into two subpopulations (Fig. 4A). One comprised DNA that migrated very slowly in both native and alkaline conditions, probably representing tails generated directly by continued elongation of the com- plementary (-) strand during its displacement. The other subpopulation contained molecules between 1 and 5 kilobases in length that were presumed to be (+) strand Okazaki frag- ments complementary to the (-) strand tails. The absence of discrete species within this subpopulation was consistent with initiation events that were random with respect to the 4x174 genomic map.

These assignments were confirmed by Southern blot anal- yses. The "P-labeled single-stranded oligonucleotide probe PstII, representing (+) strand 4x174 sequences, hybridized to the larger subpopulation but not to the Okazaki fragments

A , 2nd Alkaline-

Native - -linear mullimer

B. (-) 4x174 Products c. (+) 4x174 Products ~~

-linear multimer

,form I1 'form I11

-linear mullimer

,form II -form III

FIG. 4. Two-dimensional agarose gel electrophoretic anal- ysis of reaction products. A, "'P-labeled synthetic DNA products of a standard reaction incubated for 45 min were resolved on a two- dimensional gel as described under "Materials and Methods." Elec- trophoresis in the first dimension was under native conditions and in the second dimension under denaturing conditions. The synthetic products that migrated through these gels as diagonals underneath the form I1 and form 111 molecules were not analyzed further, since they did not appear to be immediately related to the linear multimers. B, unlabeled products of a standard reaction incubated for 90 min were similarly resolved and transferred to a nitrocellulose membrane as described under "Materials and Methods." The 32P-5'-end-labeled PstII single-stranded oligonucleotide was used to probe DNA products having (-) strand 6x174 sequences. C, after removing the hybridized probe, the Southern blot was reprobed with the 32P-5'-end-labeled PstIII single-stranded oligonucleotide to identify DNA species pos- sessing (+) strand 6x174 sequences.

DNA Helicase Activity Associated with the E. coli Primosome

(Fig. 4B). The Okazaki fragment subpopulation could be detected using the "P-labeled PstIII probe representing (-) strand 6x174 sequences (Fig. 4C). Some of the material in the larger DNA subpopulation also hybridized with this probe, implying that generation of the strand complementary to the initial single-stranded tail in the rolling-circle did not always involve synthesis of short Okazaki fragments. In addition, the original (+) strand circular templates associated with the rolling-circle molecules could be detected with the PstIII probe.

Together, these observations demonstrated that a rolling- circle product was being generated in the 6x174 SS(c) + RF DNA replication reaction. These molecules possessed multi- genome-length, double-stranded tails.

A Helicase Activity Associated with the Primosome-Since rolling-circle DNA replication requires strand displacement, which the DNA polymerase I11 holoenzyme cannot accom- plish (35, 36), the observations detailed above suggested the presence of a helicase activity associated with the primosome. To eliminate the fortuitous contamination of one of the preparations of primosomal proteins with a helicase activity, the products of DNA replication by the entire complement of 4x174 SS(c) + RF replication proteins using G4 and a-3 phage SS(c) DNAs as templates were examined. These DNAs lack a primosome assembly site and require only the DNA G primase and SSB for initiation of DNA replication. Use of the G4 and a-3 SS(c) DNAs as templates failed to support significant rolling-circle product formation (Fig. 5), indicating that there was no helicase contamination of any consequence in the protein preparations in use and that primosome assem- bly was required to generate the helicase activity.

The observations reported here and the demonstration of the intrinsic helicase activity of the DNA B protein (27) suggested that the DNA B protein was the agent responsible for the primosome-associated helicase activity. Thus the abil- ity of the general priming system (37,38), which requires only the DNA B and DNA G proteins to prime any single-stranded DNA in the absence of SSB, to support the formation of rolling-circle structures was investigated (Fig. 6A). Some large DNA molecules could be detected. However, a comparison of linear multimer formation during general priming-dependent (Fig. 6A) and primosome-dependent (Fig. 6B) DNA replica- tion indicated that, even though both systems yielded the same conversion of input SS(c) DNA templates to form I1 molecules, the latter system produced a rate of rolling-circle DNA synthesis 13-fold greater than the former system (Fig.

1 2 3 4 5

FIG. 5. Synthetic DNA products formed using other SS(c) DNA templates that lack primosome assembly sites. Standard reaction mixtures containing [a-"PJdCTP and 225 pmol of SSB- coated 6x174, G4, or a-3 SS(c) DNA templates were incubated at 30 "C. 32P-labeled synthetic DNA products were resolved by agarose gel electrophoresis performed at 1.8 V/cm for 12.5 h. Lane 1, 6x174 products formed after a 40-min incubation; lanes 2 and3, G4 synthetic products formed after 5 min and 40 min, respectively; and lanes 4 and 5, a-3 synthetic products formed after 5 min and 40 min, respectively. Prolonged incubation for up to 120 min did not result in any significant linear multimer formation from G4 or a-3 SS(c) DNA templates (data not shown).

A. 1 2 3 4 5 6 7 8 9

-"""

2307

-form I1

B. 1 2 3 4 5 6 7 8 9lO1112

""

L Y m .IO0 - lhnear rnUlIlmer

25

A"

30 60 90 120 150 180 Time (min)

FIG. 6. A comparison of the kinetics of 6x174 product for- mation with the general priming system and with the specific priming system. One hundred microliter (4 X) standard reaction mixtures containing only the DNA B (2.2 units) and DNA G (2.4 units) proteins plus pol 111 (7.2 units) in the absence of SSB ( A ) or containing SSB and all the primosomal proteins plus pol 111 ( B ) were incubated a t 30 'C. Samples were taken a t various times after the start of incubation for product analysis using agarose gel electropho- resis (2.3 V/cm for 13 h). A, lane 1, 5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 45 min; lane 5, 60 min; lane 6, 90 min; lane 7, 120 min; lane 8, 150 rnin; lane 9,180 min. B, lane 1,20 s; lane 2,40 s; lane 3 , l min; lane 4, 2 min; lane 5, 5 min; lane 6, 15 min; lane 7,30 min; lane 8, 45 min; lane 9, 60 min; lane 10, 90 min; lane 21, 120 min. C, following autoradiography, the bands in the dried gels corresponding to the form I1 and linear rnultimer DNA species were excised to quantitate radioactivity. Form I1 molecules (0-0) and linear mul- timers (W".) synthesized in the general priming system; form I1 molecules (0-0) and linear multimers (0-0) synthesized in the specific priming system.

612). Thus, these data indicate that whereas the DNA B protein is sufficient to catalyze rolling-circle DNA replication, formation of the complete primosome generates a more effi- cient helicase, probably through some modification of the DNA B helicase activity.

DISCUSSION

These studies describe the identification of a helicase activ- ity associated with the E. coli primosome as revealed through the characterization of a rolling-circle DNA replication reac- tion that occurred during 6x174 SS(c) + RF DNA synthesis. High molecular weight, double-stranded, linear, synthetic

2308 DNA Helicase Activity Associated with the E. coli Primosome

DNA products of approximately twenty 4x174 genome lengths could be detected during 4x174 SS(c) + RF DNA synthesis, consistent with the presence of a rolling-circle DNA replication mechanism. These linear multimers were com- posed of two subpopulations of DNA molecules. One consisted of extremely long molecules representing (-) strand DNA chains formed directly by strand-displacement synthesis. The other represented shorter, (+) strand Okazaki-type fragments. Similar rolling-circle structures have been observed in pre- vious studies of 4x174 replication where the primosomal proteins were present (33, 39, 40). I t is possible that the extremely high molecular weight products observed in other DNA replication systems in vitro have a similar structure. In fact, under certain conditions, the major synthetic product in the pBR322 replication system studied in this laboratory appears to be generated from rolling-circle structures (41).

A specific sequence (a primosome assembly site) on 4x174 SS(c) DNA is required for efficient primosome assembly (42). Substitution of the 6x174 DNA in the reaction mixture with either G4 or a-3 DNA, two other similar SS(c) DNA templates that lack primosome assembly sites, did not result in appre- ciable linear multimer formation. By employing the general priming system, which involves only the DNA B and DNA G proteins acting on single-stranded DNA templates not coated with SSB (37, 38), instead of the specific priming system (i.e. primosome-dependent), the production of linear multimers was greatly reduced, although still evident, under conditions that were otherwise identical.

LeBowitz and McMacken (27) have recently demonstrated that the DNA B protein is capable of acting as a helicase. The DNA B protein is a component of the primosome and may well be the protein primarily responsible for denaturing du- plex DNA strands in the activity described in this report. Previous experimental evidence has been presented that the DNA B protein functions more processively in the specific priming system than in the general priming system (43). Conceivably, this could explain why involvement of the pri- mosome results in much greater rolling-circle DNA synthesis than when the general priming system is involved, even though both systems contain the DNA B protein. The DNA B protein by itself may behave too distributively to function optimally as a helicase. In support of this, in 4x174 SS(c) + RF reactions primed with synthetic oligonucleotides and con- taining only pol I11 and a concentration of the DNA B protein optimal for specific priming, only form I1 products were made (data not shown). The nature of the conversion of the DNA B protein from its intrinsically distributive mode to the ap- parently more functional processive mode remains to be de- termined. However, it is likely that this conversion is a fundamental necessity of DNA replication in E. coli. All of the replication systems currently under study using purified proteins seem to employ a mechanism to transfer the DNA B protein to the DNA so that it can act in a processive fashion (28, 44, 45).

Two primosomal proteins, factor Y and the DNA B protein, have nucleoside triphosphatase activity. Thus, either could provide the helicase activity described here. Arai et al. (43) proposed that the ATPase activity of protein n' (factor Y) moved the primosome along the DNA. Experiments were performed in which the effects of various nonhydrolyzable analogs of ATP, GTP, and dATP on the production of mul- tigenome-length DNA molecules by isolated protein. DNA complexes known to contain at least factor Y, the DNA B protein, and the DNA polymerase I11 holoenzyme were stud- ied; however, they proved unable to differentiate clearly be- tween a DNA B- and a factor Y-driven primosomal helicase

FIG. 7. Model of rolling-circle DNA synthesis in the #X174 SS(c) 4 RF replication reaction. i, following assembly, the pri- mosome (0) migrates on the (+) strand 6x174 SS(c) template in an anti-elongation direction to synthesize primers for complementary (-) strand DNA synthesis (b) catalyzed by pol I11 (0). ii, opposite directionalities of movement on the template will result in a confron- tation between the primosome and pol I11 before complementary strand DNA synthesis is completed. Complementary strand DNA synthesis can be completed if some mechanism allows them to pass each other (iii), or if the migration of the primosome is neglible during the time it takes pol I11 to polymerize the complementary strand completely (iu). u, conversion of this form I1 DNA-protein complex to one capable of supporting rolling-circle DNA synthesis would involve transfer of the DNA B protein/primosome to the complementary strand. ui, rolling-circle DNA synthesis proceeds with the DNA B protein/primosome situated at the junction to displace the (-) strand ahead of pol I11 (and to synthesize primers for the formation of Okazaki fragments).

activity (data not shown). In fact, it appeared that if the DNA B protein or factor Y was the protein responsible for providing the helicase activity, the nucleoside triphosphate hydrolysis requirements under conditions where the polymerase and helicase activities were coupled could be different from those where helicase activity was assayed independently. Kornberg et al. (46) demonstrated this to be the case for the rep protein ATPase. There were distinct differences in K , for ATP, substrate specificity, and sensitivity to ATP analogs when the rep ATPase effector activity of single-stranded DNA was compared to that of a replicating fork.

I t now appears that the E. coli primosome may be function- ally analagous to the T7 gene 4 protein (13,14) and the phage T4 gene 41/61 proteins (15-18) in being capable of synthesiz- ing primers (15, 21-23) and acting as a helicase (19, 20). A multiprotein complex containing these functions can readily be envisaged at the replication fork to denature the parental duplex strands and to generate primers for lagging-strand DNA synthesis. The concept that the primosome moves in a direction opposite that of pol I11 (24) is consistent with the required structure-function relationships at the replication fork. In the generation of 9x174 rolling-circle molecules described here, the topology still needs further clarification (Fig. 7). At the start of the replication reaction following assembly, the primosome must reside on the viral (+) strand to synthesize primers during the conversion of SS(c) mole- cules to form I1 molecules. Subsequent formation of rolling circles requires pol I11 to continue elongation of the nascent

DNA Helicase Activity Associated with the E. coli Primosome 2309

(-) strand. If the primosome were to remain on the (+) strand, acting as a helicase, it would have to migrate in the same direction as pol 111, defying the current concept of primosome movement. One way to resolve this difficulty would be for the primosome to relocate to the nascent (-) strand at the junc- tion. In this position, it would be able to stay at the junction, moving in an anti-elongation direction, acting as a helicase and forming primers for Okazaki fragment synthesis. Relo- cation of the primosome, or its active component, would most likely be a direct intrastrand transfer. Whether and how this occurs remains to be demonstrated.

The data obtained from these studies provide evidence that there is a helicase activity associated with the E. coli primo- some. Manifestation of this helicase activity in rolling-circle formation may be an indicator of the unwinding functions operative in other reconstituted DNA replication systems as well.

Acknowledgments-We thank Dr. Jerard Hurwitz for his critical reading of this manuscript, Dr. Jonathan Minden for numerous stimulating discussions during the course of this work, and David Valentin for his technical assistance and excellent artwork.

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