Proc. Natl. Acad. Sci. USA

Vol. 74, No. 10, pp. 4190-4194, October 1977 Biochemistry

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication (DNA-dependent ATPase/multiple primers/mobile promoter/primase/phage OX174)

ROGER MCMACKEN*, KUNIHIRO UEDAt, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

Contributed by Arthur Kornberg, July 11, 1977

0.1 mM ATP, 10 mM MgCl2, and 70 Mg of bovine serum albumin per ml. Buffer B contained 120 mM MgCl2, 20 mM ATP, 40 mM spermidine-HCI, and 40 mM Tris (pH 7.5). Buffer C contained 50 mM Tris-HCl (pH 7.5), 10% (wt/vol) sucrose, 20 mM dithiothreitol, and 200 ,ug of bovine serum albumin per ml. Buffer D contained 10 mM Tris-HCI (pH 8.0), 0.1 M NaCl, and 1 mM EDTA. [y-32P]ATP was synthesized by the procedure of Maxam and Gilbert (12). The extensively purified Escherichia coli replication proteins used in this work will be described elsewhere. Antibodies against dnaB protein and protein n, and other materials and reagents, were prepared as described (3, 4). Formation and Isolation of Replication Intermediate. The replication intermediate was formed and assayed essentially as described (4). It was isolated free of unassociated protein by filtering the reaction mixture through Bio-Gel A-15m agarose (equilibrated in buffer A at 250) and collecting the void (excluded) volume. Assay of RNA Primer Synthesis. A detailed report on RNA primer synthesis on OX 174 DNA will be published elsewhere. Briefly, the components used for formation of 1.1 nmol of replication intermediate (4) were supplemented with 5 Ml of buffer B, 12 Al of buffer C, 0.8 jg of rifampicin, 140 units of primase, 4 nmol each of 3H- or a-32P-labeled CTP, GTP, and UTP, and water to 40 Ml. The mixture was incubated at 30°. Portions of the reaction mixtures were applied to Whatman DEAE-cellulose (DE81) filter paper circles and freed of unincorporated rNTPs by washing in 0.3 M ammonium formate (pH 7.8)/0.01 M sodium pyrophosphate. This assay detects oligonucleotide chains at least 4 residues long (with a triphosphate terminus) (unpublished data). In some experiments RNA primer synthesis was initiated on the isolated replication intermediate by addition of primase and appropriate rNTPs. Rifampicin had no effect on the incorporation of rNTPs in this system. Although primase can readily incorporate dNTPs into primer transcripts (10), only rNTPs were used in order to distinguish the primer from the chain elongated with DNA. Assay of DNA Replication. The reaction mixture for RNA priming described above was supplemented with 90 units of DNA polymerase III holoenzyme and 2 nmol each of 3H- or a-32P-labeled dNTPs, and incubated at 300 for 5 min. The acid-insoluble precipitate was collected to measure incorporation (13). Assay of ATPase Activity. Reaction mixtures (20 Ml) contained 10 Ml of buffer A ([.y-32P]ATP at 250 cpm/pmol), 8 Ml

ABSTRACT The first step in conversion of 4X174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. NatL Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a OX174 circle. The single dnaB protein molecule resent on each DNA circle participated in initiation of each ofthe RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation o primers for nascent (Okazaki) fragments at a replication fork.

Because replication of phage OX174 DNA in vitro relies on host replication proteins, this system has served in the assay and isolation of these proteins (1-3) and may now provide insights into their functions. Among the single-stranded (SS) phage DNAs thus far tested in replication systems in vitro, the OX174 class is unique in being converted first to a multiprotein intermediate (4, 5) to enable the synthesis of a primer by dnaG protein (primase). In contrast, primase action on DNA binding protein (DBP)-coated phage G4 DNA occurs directly, as does RNA polymerase action on DBP-coated phage M13 DNA (refs. 6-10; L. Rowen and A. Kornberg, unpublished data). In the present work, dnaB protein has been shown to be an -active component of the 4X174 replication intermediate; it sustains synthesis by primase of a ribonucleotide primer fragment when coupled to replication, and multiple primers on the OX174 circle when uncoupled from it. Our findings suggest a mechanism whereby dnaB protein utilizes its ATPase activity (11) to propel itself along the DNA strand, thus serving as a mobile promoter or recognition signal for the action of primase. MATERIALS AND METHODS Materials. Buffer A contained 10 mM imidazole-HCI (pH 7.0), 5% (wt/vol) sucrose, 20 mM KCl, 10 mM dithiothreitol,

Abbreviations: DBP, DNA binding protein; primase, dnaG protein; SS, single-stranded. * Present address: Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, 615 N. Wolfe St.,

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 accordance with 18 U. S. C. §1734 solely to indicate this fact.

Baltimore, MD 21205. t Present address: Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-Ku, Kyoto 606, Japan.

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Biochemistry: McMacken et al. Table 1. Influence of specific antibodies on formation and activity of the replication intermediate Active intermediate,* pmol After Before Antibody 155 101 Control 9 2 Anti-dnaB protein 2 1 Anti-DNA binding protein 12 105 Anti-protein i 13 91 Anti-protein n Formation of the replication intermediate and the replicative assay for determining amount of active intermediate were performed as described (4). * Intermediate formation was measured before or after addition of gamma globulin (10-20 Mg).

of buffer C, 500 pmol of OX174 SS DNA, 2 ug of DBP, and the protein fraction to be assayed. Reaction mixtures were incubated at 300 and the conversion of 32p to a form not adsorbed to Norit was measured. Other Methods. Polyacrylamide gel electrophoresis in 7 M urea and subsequent autoradiography were performed as described (14). RESULTS OX174 Replication Intermediate Contains dnaB Protein. In a previous study (4), the intermediate produced by interaction of OX174 DNA with five bacterial replication proteins was judged to contain DBP and possibly dnaB and dnaC proteins, but not proteins i and n. The amount of intermediate formed was directly proportional to the amount of dnaB added, about one molecule per circle (4). With anti-dnaB gamma globulin, which neutralizes dnaB protein activity, we have observed strong inhibition of both formation and activity of the OX 174 replication intermediate (Table 1). The replicative activity of the isolated intermediate was more than 97% inhibited by anti-dnaB gamma globulin. The use of labeled dnaB protein has provided additional evidence for its presence in the intermediate (unpublished data). Failure of gamma globulin fractions directed against proteins i and n to impair replicative activity of the intermediate, once formed, is consistent with earlier indications that these proteins are catalytic in function (4). The precise role of dnaC protein remains to be clarified. Physical Association of dnaB ATPase with Replication Intermediate. The DNA-dependent ATPase activity of dnaB protein (11) was found in the replication intermediate fraction prepared by agarose gel filtration.J That dnaB protein in the intermediate is the source of ATPase activity is indicated by neutralization of over 70% of the activity in the excluded gel fraction by anti-dnaB gamma globulin; antibody against protein i had no effect. RNA Primer Synthesis Dependent on Primase Occurs Only on Replication Intermediate Form of OX174 DNA. The five E. coh replication proteins required for synthesis of the replication intermediate complex and primase are necessary for synthesis of RNA transcripts on OX174 viral DNA in the presence of the four rNTPs (10). Inasmuch as the 4X174 replication intermediate is synthesized before primase acts in replication of DNA (2, 4), it seems probable that this primer-synthesizing * ATPase

activity associated with the replication intermediate (600 pmol of ATP hydrolyzed per mi per pmol of kX174 circles) agrees with the DNA-dependent ATPase activity expected for a single dnaB protein molecule bound to each OX174 DNA molecule (660 pmol of ATP hydrolyzed per min per pmolof-daB-protein).

Proc. Nati. Acad. Sci. USA 74 (1977)

4191

Table 2. Requirements for RNA synthesis on X174 DNA Antiprotein i rNMP gamma incorpoProteins Viral Primase globulin ration* added template 0 + DBP 4X174 + 26 DBP G4 DBP, dnaB, OX174 1 dnaC, i, n DBP, dnaB, kX174 210 + dnaC, i,n DBP, dnaB, kX174 4 + + dnaC, i,n Replication 0 intermediate None Replication 180 + intermediate None Replication + 165 + intermediate None RNA primer was synthesized as in Materials and Methods except that replication proteins, 650 pmol of isolated replication intermediate, and 12 ,g of anti-protein i gamma globulin were added as indicated. Reaction mixtures with antibody were incubated at 0° for 10 min prior to incubation at 300. * rNMP per circle.

protein can use only this form of kX174 DNA. Although primase readily transcribes DBP-coated phage G4 DNA, it is inert on DBP-coated 4X174 DNA (Table 2). RNA synthesis dependent on primase is observed on OX174 DNA only when dnaB protein, dnaC protein, and proteins i and n are also present (Table 2). The replication intermediate, isolated from unassociated protein, has the form of 4X174 DNA required for transcription by primase (Table 2). Antibody against protein i directly blocks formation of the replication intermediate (4), but has no effect on priming capacity or the replicative activity of intermediate once it is formed (Table 2). Synthesis of Multiple Primers on 4iX174 DNA in Absence of DNA Replication. The magnitude of RNA priming on kX174 DNA was surprisingly large. Whereas primase action on phage G4 DNA polymerizes 25-30 ribonucleotides per circle (15) (Table 2), 5-10 times as many were incorporated with ckX174 DNA as template (Table 2). Electrophoresis of isolated kX174 RNA products (Fig. 1) indicated that most of the recovered RNA product was 15-30 nucleotides long. Fingerprint analysis of this OX174 primer RNA, after digestion with T1 RNase or RNase A, yielded a pattern of sequence complexity comparable to that of RNA several hundred nucleotides long (unpublished data). To determine whether the 4X174 RNA oligonucleotides were breakdown products of a much longer RNA chain or if each had been initiated de novo, isolated OX174 replication intermediate was transcribed in the presence of [y-32P]ATP and 3H-labeled CTP, GTP, and UTP. Approximately one ['y-32P]ATP was incorporated per oligonucleotide (16-26 ribonucleotides long) (Table 3). Electrophoresis of the 4X174 priming reaction mixture directly on polyacrylamide in 7 M urea confirmed that ['y-32P]ATP incorporation dependent on primase formed short RNA fragments with chain lengths of 6 to more than 40 residues (unpublished data). The rough correspondence in the ratio of total ribonucleotide to [y-32P]ATP (Table 3) with the chain length of the average RNA product suggests that each kX174 RNA transcript on a OX174 circle is initiated at its 5' terminus with ATP. dnaB Protein Is Continuously Required for RNA Synthesis on ikX174 DNA. The single dnaB protein molecule in the

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Biochemistry: McMacken et al. A

tRNA

B RNA primers

Markers

Proc. Nati. Acad. Sci. USA 74 (1977) Table 3. Incorporation of [y-32P]ATP into 4X174 RNA primers Exp. 2 Exp. 1

[3H]rNMP incorporated, residues/circle

158

131

[y-32P]ATP incorporated,

.-

80

-0

XC(58)

-*-

33

-_- 26

18 --8-

8 6 residues/circle 16 26 rNMP/ATP Isolated intermediate (900 pmol in 30 Mil of buffer A containing [y-32P]ATP at 32,000 cpm/pmol) was incubated with 5 Ad of buffer C, 115 units of primase, 5 nmol each of 3H-labeled CTP, GTP, and UTP (2000 cpm/pmol), and H20 (to 50 Ml) for 20 min at 300. Total rNMP and [-y-32P]ATP incorporation were measured by the DEAE-cellulose paper assay of aliquots taken before and after incubation.

F). The rapid conversion of circular template to almost completely duplex DNA which follows synthesis of the initial primer appears to preclude synthesis of additional primer transcripts on the same DNA molecule. However, when multiple primers were synthesized on a circle before DNA replication was initiated, much shorter DNA chains were also produced (median length approximately 200 residues, slot D). Thus, the multiple RNA primers were not synthesized at random sites but rather at relatively uniform intervals on the template. This interprimer distance appears to be inversely related to concentration of primase (slots C and H). Continual migration of the dnaB protein along the template strand to allow transcription to be initiated at a series of sites could account for such a pattern of priming.

-----BPB (13)

FIG. 1. Gel electrophoresis of 4X174 RNA primers. RNA primer synthesis on 4X174 DNA was for 10 min at 300 with 32P-labeled rNTPs at 2 X 104 cpm/pmol; 81 ribonucleotide residues were incorporated per input circle. The reaction mixture was filtered through Bio-Gel A-15m agarose equilibrated in buffer D; RNA stably hybridized to template DNA in the void volume (215 nucleotides long) was precipitated with ethanol. RNA samples were fractionated by electrophoresis in 12% polyacrylamide gel containing 7 M urea. The approximate length in nucleotides is given. (A) 32P-Labeled Drosophila tRNA; (B) 4X174 RNA primers. XC, xylene cyanol; BPB, bromphenol blue.

4X174 replication intermediate is required not only for initiation of primase action, but for its capacity to sustain the synthesis of many RNA primers produced in the absence of DNA replication. Addition of anti-dnaB gamma globulin any time after RNA synthesis had been initiated halted it abruptly (Fig. 2). Similar results were obtained when incorporation of [y32P]ATP into RNA was measured (data not shown). Continued availability of primed DNA for replication after antibody treatment indicated that precipitation of the DNA by anti-dnaB gamma globulin was not the cause of these inhibitions (unpublished data). These results suggest that persistence of active dnaB protein is required for initiation of every RNA chain on

DISCUSSION The rate-limiting step in in vitro replication of 4X174 SS, circular DNA to the duplex form is conversion of the viral strand to a nucleoprotein complex, the replication intermediate (2, 4). Formation of this intermediate entails binding to the DNA circle (coated by binding protein) of a single dnaB protein molecule. (The roles of three other proteins, namely, dnaC protein and proteins i and n, absolutely essential for this binding, have yet to be clarified.) Synthesis of primer RNA by primase occurs only on DNA in the activated intermediate form; this transcription, when uncoupled from DNA synthesis, is extensive, and multiple primers are made on each DNA circle. Our results suggest that the single dnaB protein molecule bound in the replication intermediate participates in de novo initiation of these oligonucleotide primers. From the size of the DNA

OX174 DNA.

Arrangement of Multiple RNA Primers on 4X174 DNA Template. The relative positions of the RNA primers on the viral strand were assessed from the lengths of the DNA chains after DNA polymerase III holoenzyme extended the primers. From the sizes of the DNA chains made at various levels of RNA priming, the spacing of the primers on the template could be deduced. In coupled RNA priming-DNA replication reaction, the product DNA chains were primarily the size of full-length linear 4X174 DNA, as judged by electrophoretic mobility in polyacrylamide gel under denaturing conditions (Fig. 3, slot

Time of incubation, min FIG. 2. Effect of anti-dnaB antibody on ongoing +X174 RNA primer synthesis. Isolated OX174 replication intermediate (600 pmol) was transcribed with primase using 3H-labeled rNTPs (2000 cpm/ pmol). At various times (arrows) after RNA primer synthesis had been initiated, 24 Mg of anti-dnaB or anti-protein i gamma globulin was added to the reaction mixture; incubation was continued until total incubation time was 20 min. rNTP incorporation was measured by DEAE-cellulose paper assay. Time course of RNA synthesis in absence of antibody is the control.

McMacken et al. BMProc. Natl. Acad. Sci. USA 74 (1977) Biochemistry: * 32P ^ Priming, min Markers 4OX circle * OX linear

1-DNA-DNA I-RNA0 20 1.5 20 - 0 20 20

A B

C

D

E

G

F

4193

H

_w - 1465

_wU

tInl

-

820

v 550 330

220 165

\5 > 5~~~~~~~~~~~~~~ ~~~~~~Ligase5 agging

Leading

strand

strand

FIG. 4. Hypothetical scheme for mechanism of dnaB protein action at a replication fork in E. coli chromosome. See text for details.

4147 S_,

v10

FIG. 3. Size of complementary strand 4X174 DNA synthesized after priming with various numbers of RNA transcripts per circle. Isolated replication intermediate was transcribed with primase in the presence of 32P-labeled rNTPs (4000 cpm/pmol; slots B-D) or 3Hlabeled rNTPs (2000 cpm/pmol; slots A and F-H) or one-sixth the primase (20 units/nmol of intermediate; slot H). After times shown, holoenzyme and no dNTPs (slot B) or 3H-labeled dNTPs (slots C and D) or 32P-labeled dNTPs (slots A and F-H) were added; the mixtures were incubated 5 min longer. The sizes of the DNA products, after heat denaturation in 0.5 M formaldehyde, were determined in a 2.5-7.5% gradient polyacrylamide gel containing 7 M urea. In the priming reactions for the samples applied to slots B, C, D, G, and H, incorporation of rNMP residues per input 4X174 circles were, respectively, 226, 20, 226, 114, and 49. Slot A, 4X174 replicative form II (not heat denatured); slot E, 32P-labeled 4X174 DNA and d(pApG)5; slot I, 32P-labeled simian virus 40 fragments from a Hae III restriction endonuclease digest.

chains synthesized when the gaps between the primers are subsequently filled by DNA polymerase action, it appears that at high concentrations of primase the multiple primers are spaced at regular intervals in the range of 70-500 nucleotides along the template strand. If dnaB protein, once bound, can propel itself along a DNA strand, then a single molecule can participate in initiating primer chains at many different sites on the template. Thus, conversion of OX 174 SS DNA to the duplex might occur by this sequence of steps: (i) dnaB protein is transferred to kX174 DNA (coated with DNA binding protein) by the action of dnaC protein, proteins i and n, ATP, and Mg2+, forming the replication intermediate; binding to DNA may be at a specific location. (ii) dnaB protein migrates processively on the DNA, utilizing ATP to propel itself. (iii) Recognition of bound dnaB protein by primase initiates synthesis of an RNA transcript at or near that point. (iv) dnaB protein, remaining on the DNA, moves along the strand to a nearby site (approximately 200 nucleotides away under our in vitro conditions) where synthesis of another primer is initiated by primase. Polarity of movement is presumably in a direction opposite to growth of the primer and therefore 5' -- 3' on the SS template. Uncoupled from replication, this sequence is repeated many times over. With DNA polymerase III holoenzyme and deoxynucleoside tri-

phosphates present, the first primer is very rapidly elongated into a full-length, linear complementary strand. This reconstituted 4X174 replication system appears to be similar to that used by the cell for initiating nascent DNA fragments (Okazaki fragments) at a replicating fork in its own chromosome (2, 3, 16-19). It is clear from both in vivo and in vitro studies that dnaB protein, in particular, acts at replication forks of the chromosome (16, 20-22). Its function in host replication may be similar to that in replication of 6X174 DNA in vitro. In such a mechanism (Fig. 4) we speculate that the dnaB protein, once transferred by initiation proteins to the bacterial chromosome, migrates processively on the newly exposed template strand behind the replicating fork in 5' -- 3' direction of that strand. The dnaB protein, operating at or near the moving replication fork, serves as a "mobile promoter" for primase-dependent synthesis of the primer transcripts for the Okazaki fragments. By circumventing the need to transfer a new dnaB protein molecule to the template strand for synthesis Origin 53

i1111.1111111111111111115' 3'J51I1111111 1 ) Unwinding at origin 2) Binding of dnaB protein

5

B

3

1 ) Priming

2) Replication Leading

Lagging

B

5' 3' B

ILaggh RNA primer

FIG. 5. Model for role of dnaB protein in priming bidirectional DNA replication at chromosomal origin of replication. Origin is origin of DNA replication; B is dnaB protein. See text for details.

4194

Biochemistry: McMacken et al.

of each nascent fragment, the slow step of forming the replication intermediate can be avoided. The mechanism proposed for dnaB protein action may be applicable in the initiation of bidirectional replication at the chromosomal origin (Fig. 5), as an alternative to the mechanism involving duplex OX174 DNA in which a specific nicking action initiates covalent extension of the leading strand (23, 24). A dnaB protein molecule is bound on each strand at the replication origin and moves in 5' -- 3' direction. Thus, each molecule would direct priming both of the continuously synthesized strand (the single initiating event for the leading strand) and of the nascent fragments of the discontinuously synthesized strand (multiple events for the lagging strand). In this scheme, specificity for initiating a round of replication (where dnaB protein is used) would reside in proteins that transfer dnaB to DNA at the chromosomal origin. As an example, the replication protein P of bacteriophage X, a protein believed to interact with dnaB protein (25), may specifically transfer dnaB to the origin of replication on X DNA. We thank Dr. Bik Tye, Mariana Wolfner, and Dr. Kirk Fry, all of this department, and Dr. P. Englund of The Johns Hopkins University for 32P-labeled DNA and RNA markers. A portion of this investigation was completed at The Johns Hopkins University (by R.M.) and was supported by Grant FR-05445 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health. This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. A grant from the Eleanor Roosevelt International Union Against Cancer provided postdoctoral fellowship support for K.U. Grants from the National Institutes of Health and the Kaiser Foundation provided postdoctoral fellowship support for R.M. 1. Schekman, R., Wickner, W., Westergaard, O., Brutlag, D., Geider, K., Bertsch, L. & Kornberg, A. (1972) Proc. Natl. Acad. Sci. USA 69,2691-2696. 2. Wickner, S. & Hurwitz, J. (1974) Proc. Natl. Acad. Sci. USA 71,

4120-4124. 3. Schekman, R., Weiner, J. H., Weiner, A. & Kornberg, A. (1975) J. Biol. Chem. 250,5859-5865. 4. Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Nati. Acad. Sci. USA 73, 752-756.

Proc. Natl. Acad. Sci. USA 74 (1977) 5. Wickner, S. & Hurwitz, J. (1975) in DNA Spsthesis and Its Regulation, eds. Goulian, M. M., Hanawalt, P. C. & Fox, C. F. (W. A. Benjamin, Inc., Menlo Park, CA), pp. 227-238. 6. Westergaard, O., Brutlag, D. & Kornberg, A. (1973) J. Biol. Chem. 248, 1361-1364. 7. Wickner, W., Brutlag, D., Schekman, R. & Kornberg, A. (1972) Proc. Nati. Acad. Sci. USA 69,965-969. 8. Geider, K. & Kornberg, A. (1974) J. Biol. Chem. 249, 39994005. 9. Bouche, J.-P., Zechel, K. & Kornberg, A. (1975) J. Biol. Chem. 250,5995-6001. 10. McMacken, R., Bouche, J.-P., Rowen, S. L., Weiner, J. H., Ueda, K., Thelander, L., McHenry, C. & Kornberg, A. (1977) in Nucleic Acid-Protein Recognition, ed. Vogel, H. J. (Academic Press, Inc., New York), pp. 15-29. 11. Wickner, S., Wright, M. & Hurwitz, J. (1974) Proc. Natl. Acad. Sci. USA 71,783-787. 12. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,560-564. 13. Weiner, J. H., Bertsch, L. L. & Kornberg, A. (1975) J. Biol. Chem. 250, 1972-1980. 14. Maniatis, T., Jeffrey, A. & Van de Sande, H. (1975) Biochemistry 14,3787-3794. 15. Rowen, S. L. & Bouche, J.-P. (1976) Fed. Proc. 35, abstr. 1418. 16. Wechsler, J. A. & Gross, J. D. (1971) Mol. Gen. Genet. 113, 273-284. 17. Filip, C. C., Allen, J. S., Gustafson, R. A., Allen, R. G. & Walker, J. R. (1974) J. Bacteriol. 119,443-449. 18. Wickner, S. & Hurwitz, J. (1976) Proc. Natl. Acad. Sci. USA 73, 1053-1057. 19. McHenry, C. S. (1976) Fed. Proc. 35, abstr. 1840. 20. Schaller, H., Otto, B., Nusslein, V., Huf, J., Herrmann, R. & Bonhoeffer, F. (1972) J. Mol. Biol. 63, 183-200. 21. Wechsler, J. A., Nusslein, V., Otto, B., Klein, A., Bonhoeffer, F., Herrmann, R., Gloger, L. & Schaller, H. (1973) J. Bacteriol. 113, 1381-1386. 22. Nusslein, V., Henke, S. & Johnston, L. H. (1976) Mol. Gen. Genet. 145, 183-190. 23. Scott, J. F., Eisenberg, S., Bertsch, L. L. & Kornberg, A. (1977) Proc. Natl. Acad. Sci. USA 74, 193-197. 24. Eisenberg, S., Griffith, J. & Kornberg, A. (1977) Proc. Natl. Acad. Sci. USA 74,3198-3202. 25. Georgopoulos, C. P. & Herskowitz, I. (1971) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 553-564.