©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Protects in the Leading-strand Polymerase Complex at the Replication Fork (*)

(Received for publication, October 5, 1995; and in revised form, December 20, 1995)

Sungsub Kim (1) H. Garry Dallmann (2) Charles S. McHenry (2) Kenneth J. Marians (1) (3)

From the  (1)Graduate Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021, the (2)Department of Biochemistry, Biophysics, and Genetics, and the Graduate Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the (3)Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Replication forks formed in the absence of the subunit of the DNA polymerase III holoenzyme produce shorter leading and lagging strands than when is present. We show that one reason for this is that in the absence of , but in the presence of the -complex, leading-strand synthesis is no longer highly processive. In the absence of , the size of the leading strand becomes proportional to the concentration of beta and inversely proportional to the concentration of the -complex. In addition, the beta in the leading-strand complex is no longer resistant to challenge by either anti-beta antibodies or poly(dA):oligo(dT). Thus, is required to cement a processive leading-strand complex, presumably by preventing removal of beta catalyzed by the -complex.


INTRODUCTION

The replication fork of Escherichia coli is extraordinarily processive. Two replication forks form at oriC and synthesize roughly 2.2 megabase pairs of DNA before they meet again in the terminus region. The enzymatic machinery at the replication fork accomplishes this task while supporting two distinct modes of DNA synthesis. Whereas the leading strand is synthesized in a continuous fashion that reflects the overall processivity of the replication fork, the lagging strand is synthesized discontinuously in short Okazaki fragments 2 kb (^1)in length (1) . This issue is compounded by the fact that the mechanism responsible for maintaining the processivity of the replicative polymerase on either strand is the same, a complex between the beta subunit and the polymerase core of the DNA polymerase III holoenzyme (Pol III HE)(2, 3) .

The core(4) , the catalytic polymerase/exonuclease subassembly of the Pol III HE, is essentially a distributive enzyme, synthesizing only a few nucleotides per primer binding event(5) . For conversion to a processive enzyme, the core must be clamped onto DNA by associating with the beta subunit(2, 3) , a dimer that encircles double-stranded DNA (6, 7) . beta can be loaded onto the primer terminus via the action of five other HE subunits that themselves associate, forming the -complex (, , `, , and )(8) .

Using rolling circle DNA replication supported by a tailed form II DNA template (TFII) and the X-type primosomal proteins (PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG), the single-stranded DNA-binding protein (SSB), and either bona fide Pol III HE or HE reconstituted from purified subunits, we have reconstituted the coordinated leading- and lagging-strand synthesis of the E. coli replication fork(9, 10, 11, 12, 13) . These replication forks were shown to have processivities of at least 0.5 megabase, yet they also made Okazaki fragments whose average length was about 1.8 kb(9) . This suggested that the required protein elements for establishing processivity and triggering lagging-strand polymerase cycling were present.

The mechanism for inducing polymerase release on the lagging strand is yet to be established firmly. We have proposed that protein-protein interactions between a primase synthesizing the primer for the next Okazaki fragment and the lagging-strand polymerase initiates termination of synthesis and keys recycling of the polymerase to the new primer(11, 13) . In the bacteriophage T4 system, Hacker and Alberts (14) have argued that the lagging-strand polymerase will dissociate spontaneously from the gene 45 protein clamp when it hits the 5`-end of the previous Okazaki fragment. Stukenberg et al.(15) have made similar arguments for the E. coli fork.

Challenge experiments have shown that both the polymerase and helicase on the leading-strand side of the fork are processive(16) . Our recent studies have demonstrated that a protein-protein interaction between the subunit of the Pol III HE and DnaB is required to mediate rapid replication fork movement. (^2)We show here that contributes in yet another way to processivity on the leading strand by protecting beta in the leading-strand polymerase complex from being removed by the action of the -complex.


MATERIALS AND METHODS

Reagents, DNAs, and Enzymes

NTPs, dNTPs, and poly(dA) were from Pharmacia Biotech Inc. [alpha-P]dATP was from Amersham, Bio-Gel A-150m was from Bio-Rad. Oligo(dT) was synthesized using an Applied Biosystems 380A DNA Synthesizer and was used after gel purification. Alkaline phosphatase was from Boehringer Mannheim. DNAs from bacteriophages flAY-7M and flR229-A/33 were prepared as described previously(17) .

Replication Proteins

SSB was prepared according to Minden and Marians(18) . PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG were prepared according to Marians(19) . Pol III HE subunits and subassemblies were prepared as follows: core (4) and to be described, (^3)beta (20) , and (21) , and ` to be described, (^4)and (22) .

Rolling Circle DNA Replication

TFII DNA template was prepared as described by Mok and Marians (16) using [^3H]TTP. Rolling circle reaction mixtures (12 µl final volume) containing 50 mM Hepes-KOH (pH 7.9), 12 mM MgOAc, 10 mM dithiothreitol, 5 µM ATP, 80 mM KCl, 0.1 mg/ml bovine serum albumin, 1.1 µM SSB, 0.42 nM TFII, 3.2 nM DnaB, 56 nM DnaC, 680 nM DnaG (present only in the experiment shown in Fig. 1), 28 nM DnaT, 2.5 nM PriA, 2.5 nM PriB, 2.5 nM PriC, and HE subunits and subassemblies (formed by mixing purified subunits at equimolar ratios) at 28 nM unless indicated otherwise, were incubated at 30 °C for 2 min. ATP was then added to 1 mM along with GTP, CTP, and UTP each to 200 µM and the dNTPs to 40 µM, and the incubation continued for 1.5 min. [alpha-P]dATP (to 2000-4000 cpm/pmol) was then added and the incubation continued for 10 min. The reaction was terminated by the addition of EDTA to 40 mM. Total DNA synthesis was determined by acid-precipitating an aliquot of the reaction mixture, and the DNA products were analyzed by alkaline agarose gel electrophoresis as described previously(9) .


Figure 1: Replication forks formed in the absence of synthesize shorter leading and lagging strands. Standard rolling circle replication reactions in the presence of primase and either in the presence or absence of as indicated were performed, processed, and analyzed as described under ``Materials and Methods.'' Total DNA synthesis in the reactions shown here were 186 pmol + and 19 pmol - of [alpha-P]dAMP incorporated into acid-insoluble product.



Formation of beta-TFII DNA Complexes

A reaction mixture (120 µl) containing 50 mM Hepes-KOH (pH 7.9), 12 mM MgOAc, 10 mM dithiothreitol, 2 mM ATP, 80 mM KCl, 0.1 mg/ml bovine serum albumin, 200 µM each of CTP, UTP, and GTP, 40 µM dNTPs, 0.83 nM TFII, 82 nM beta, 41 nM -complex, and 1.1 µM SSB, was incubated at 30 °C for 10 min. This mixture was then filtered at room temperature through a 5-ml Bio-Gel A-150m column formed in a Falcon 5-ml pipette equilibrated and developed with a buffer containing 62.5 mM Hepes-KOH, pH 7.9, 15 mM MgOAc, 12.5 mM dithiothreitol, 125 µg/ml bovine serum albumin, 100 mM KCl, 6.25 µM ATP, and 50 µM each of dCTP and dGTP. Fractions (200 µl) were collected, and excluded material was located by identifying the presence of the [^3H]TFII.

Processivity Challenge Experiments

Poly(dA):Oligo(dT)

Poly(dA):oligo(dT) was prepared by annealing 7 µM poly(dA) with 8.6 µM oligo(dT) in a reaction mixture (30 µl) containing 50 mM Tris-OAc (pH 7.5), 200 mM NaCl, and 20 mM MgCl(2) at 75 °C for 2 min followed by slow cooling (2 h) to room temperature. A mixture (2 µl) of the preprimosomal proteins (the primosomal proteins in the absence of primase), SSB, core, and -complex, either in the presence or absence of , was added to 20 µl of the peak fraction of the beta-TFII DNA complex to give their standard reaction concentrations. The reaction was initiated by the addition of ATP to 1 mM, GTP, CTP, and UTP each to 200 µM, and dATP and dTTP each to 40 µM, and incubated at 30 °C for 1.5 min. [alpha-P]dATP and poly(dA):oligo(dT) (to a final concentration of 120 nM 3`-ends), as indicated, were then added, and the reaction continued for 10 min at 30 °C. Reactions were terminated by the addition of EDTA to 40 mM and processed and analyzed as described above.

Anti-beta Antibody

Standard rolling circle reaction mixtures were assembled either in the presence or absence of . The effect of the anti-beta antibody on initiation was assessed by including it in the reaction mixture from the start, before the ATP concentration was raised and before the dNTPs and other NTPs were added. The effect of the antibody on elongation was determined by adding the antibody along with the [alpha-P]dATP, 1.5 min after the reactions had been initiated. Reactions were processed and analyzed as described above.


RESULTS

Replication Forks Formed in the Absence of Synthesize Shorter Leading and Lagging Strands

In the course of our studies on the roles of the subunits of the Pol III HE at the replication fork, we noted that replication forks formed in the absence of synthesized shorter leading and lagging strands than those reconstituted with (Fig. 1). We found that played a central role in ensuring the generation of a processive replication fork. A protein-protein interaction between and the replication fork helicase DnaB was required to mediate rapid fork movement^2 and, as described here, in the absence of , the leading-strand side of the fork becomes nonprocessive.

The processivity of the E. coli replication fork in vitro is reflected in its high rate of speed(9, 16) , the inaccessibility of beta on the leading strand to challenge(16, 23) , and the insensitivity of the length of the leading strand to the concentration of beta(9) . Forks that lacked and free -complex were still processive.^2 We examined whether this held true in the presence of added -complex as well.

The length of the leading strand synthesized by -less replication forks was dependent on the concentration of the beta subunit (Fig. 2). This is strikingly different from the situation in the presence of where, although overall DNA synthesis is dependent on beta, the size of the leading strand is independent of the beta concentration(9) . This is consistent with the leading-strand beta-core complex needing to form only once for synthesis of a long continuous DNA product. This observation suggested that multiple betas were required in the absence of in order to synthesize the leading strand. That is, beta was being cycled in and out of the leading-strand complex.


Figure 2: The concentration of beta affects the size of the leading strand at -less replication forks. Standard rolling circle replication reactions performed in the absence of and primase and containing the indicated concentrations of beta were processed and analyzed as described under ``Materials and Methods.'' 5.0 pmol, 5.2 pmol, 5.6 pmol, and 5.3 pmol of [alpha-P]dAMP were incorporated into acid-insoluble product in the reactions shown from left to right. The DNA products with electrophoretic mobilities near the 6.4-kb marker and just slower than the 9.4-kb marker result from the addition of a limited number of nucleotides to the TFII DNA template and to dimer TFII, respectively.



The only group of proteins available in these reactions that could affect beta loading onto 3`-ends was the -complex. It followed that it was the -complex that was removing beta from the leading-strand complex as well. If this were true, then the size of the leading strand synthesized in the absence of should also be affected by the concentration of the -complex. This proved to be the case.

In the presence of , the concentration of the -complex had no effect on leading-strand synthesis (Fig. 3A), whereas in the absence of , the size of the leading strand synthesized decreased progressively as the concentration of -complex increased (Fig. 3B). At the highest concentrations of -complex tested, DNA synthesis was significantly inhibited. These observations suggested that, in the absence of , beta was being cycled on and off the leading strand by the action of the -complex.


Figure 3: The size of the leading strand synthesized by -less replication forks is inversely proportional to the concentration of the -complex. Standard rolling circle replication reactions in the absence of primase and either in the presence (A) or absence (B) of and containing the indicated concentration of -complex were performed, processed, and analyzed as described under ``Materials and Methods.'' Total DNA synthesis in the reactions shown here were: 19.8, 37.4, 37.9, 36.1, and 24.3 pmol in A and 3.6, 4.7, 4.9, 6.0, and 5.1 pmol in B from left to right, respectively.



Replication Forks Formed in the Absence of Are Nonprocessive

We used two types of challenges in order to test the processivity of replication forks formed in the absence of : anti-beta antibody and poly(dA):oligo(dT). The former is, of course, specific for beta, whereas the latter serves to capture beta, core, and probably DnaB, all of which are normally processive on the leading strand(9, 16) .

Replication forks capable of synthesizing leading strands were formed using a beta-TFII DNA complex, the preprimosomal proteins (the primosomal proteins minus DnaG), SSB, core, and the -complex either in the presence or absence of . After 1.5 min, the poly(dA):oligo(dT) challenge was added. Forks formed in the presence of were completely resistant to the challenge (Fig. 4). On the other hand, those formed in the absence of were sensitive, as evidenced by the sharply reduced size of the leading-strand product (Fig. 4). This demonstrated that, in the absence of , at least one of the normally processive enzymatic components on the leading strand was now acting distributively. The data described above suggested that this component was beta. To assess this, we repeated the challenge experiment using anti-beta antibody.


Figure 4: -less replication forks are distributive. -less replication forks were reconstituted using the beta-TFII DNA complex, the preprimosomal proteins (PPP) (the primosomal proteins in the absence of primase), SSB, core, , and the -complex as indicated. 1.5 min after replication was initiated, poly(dA):oligo(dT) was added as indicated and the reactions continued for another 10 min. DNA products were processed and analyzed as described under ``Materials and Methods.''



beta becomes inaccessible to anti-beta antibody once it forms an initiation complex with core(24) . We used this observation previously to show that the beta on the leading strand was processive, i.e. it was insensitive to the presence of the antibody (16) . We repeated those experiments here using standard rolling circle replication reactions reconstituted in either the absence or presence of (Fig. 5). In each case, replication was inhibited if the anti-beta antibody was added before initiation. However, if the anti-beta antibody was added 1.5 min after initiation, only replication forks formed in the presence of were resistant, those formed in its absence were inhibited almost completely.


Figure 5: beta on the leading strand of -less replication forks is sensitive to anti-beta antibody. Standard rolling circle replication reactions were assembled as indicated in the absence of primase and in either the absence or presence of . In the lanes labeled with an I, anti-beta antibody was added before beta to the reaction mixtures. In the lanes labeled with an E, anti-beta antibody was added 1.5 min after initiation of DNA synthesis, as indicated under ``Materials and Methods.'' The distinct bands that appear at about 7 and 14 kb are inactive monomer and dimer templates that become labeled by the addition of a few nucleotide residues.



These data show that -less replication forks are nonprocessive because the beta in the leading-strand complex has now become vulnerable to disassembly catalyzed by the -complex.


DISCUSSION

During DNA replication, the processivity of the replication fork arises from two contributing sources: the DNA helicase and the leading-strand polymerase. We have recently shown that rapid fork movement requires a physical connection between the polymerase and the helicase that is mediated by a -DnaB protein-protein interaction.^2 In the absence of this interaction, the polymerase follows behind the helicase at a rate equal to the slow (ca. 40 nt/s) unwinding rate of the helicase alone, whereas upon establishing a -DnaB contact, DnaB becomes a more effective helicase, increasing its translocation rate by more than ten-fold. We show here that also contributes directly to the processivity of the leading-strand polymerase by preventing the -complex-catalyzed removal of beta from the leading strand.

In the absence of , the length of the leading strand was dependent on the concentration of beta and inversely proportional to the concentration of the -complex. In the presence of , the concentrations of these subunits had no effect on leading-strand synthesis(9) . This suggested that at a -less replication fork, beta, and probably core, were being reused to synthesize the leading strand in short bursts. The distributive nature of the -less replication fork was demonstrated directly in challenge experiments. Thus, not only is essential for maintaining a high rate of fork movement,^2 it is also essential for maintaining the leading-strand beta-core complex in a form that is resistant to the action of the -complex.

This suggests that, at least on the leading strand, may contact beta, perhaps covering a face of the protein that is essential for interaction with the -complex. This is consistent with the genetic finding that mutations in dnaX (encoding both and ) can be suppressed by mutations in dnaN (encoding beta)(24) . Our recent studies show that the C-terminal domain of alpha binds both and beta, thus placing them in proximity and presumably permitting direct protection.^3 Alternatively, contacting core (probably via alpha) causes a rearrangement of the beta-core complex.

It is not clear when, at a -less replication fork, beta becomes available for attack by the -complex. In the absence of both and the -complex, the leading-strand beta-core assembly is processive for at least 20 kb, following along on the DNA behind the slow moving helicase.^2 It has also been shown that the combination of beta and core will synthesize full-length product in a single binding event on poly(dA):oligo(dT)(5) . Perhaps the -less leading-strand polymerase moves fitfully behind the helicase, occasionally stalling, and occasionally jumping ahead, and it is while the polymerase is stalled that beta can be targeted by the -complex for recycling. On the other hand, in the absence of , beta may be bound less tightly to the pol III core, occasionally dissociating and diffusing away unidirectionally. Reassociation with the core would be rapid because of the constraints imposed by diffusion in two-dimensional space along the DNA fiber. It may be that beta is removed by -complex while diffusing on the DNA.

In solution, dimerizes the core to give Pol III`(25) . If this structure is similar at the fork, then the core-beta complex on the lagging strand should be affected by in a similar fashion as the core-beta complex on the leading strand. Thus, the lagging-strand polymerase should be refractory to recycling. Yet, this is clearly not the case. The lagging-strand core has been shown to cycle from the just-completed Okazaki fragment to the next primer terminus(12, 13) . It seems likely, then, that mechanisms exist on the lagging strand that override the protective effect of .

These mechanisms could take one of many forms. Conformational rearrangements could occur upon termination of Okazaki fragment synthesis, by whatever mechanism, that expose the lagging-strand core-beta to disassembly by the -complex. On the other hand, it may be that the protective effect of is specific to the leading-strand polymerase.

Stukenberg et al.(15) demonstrated that beta would dissociate from core when the polymerase collided with the 5`-end of a DNA strand bound to a template, a situation similar to the lagging-strand polymerase colliding with the 5`-triphosphate end of the primer on the previous Okazaki fragment. These experiments were conducted using Pol III* and beta on gapped duplex bacteriophage RF DNA. was present, yet disassembly of the core-beta complex occurred. Thus, does not prevent recycling. This suggests that, in solution, and when a dimeric HE subassembly binds a primer end, the polymerase bound to the primer cannot sense, a priori, whether it should be a leading- or a lagging-strand polymerase. Functional asymmetry that defines the leading- and lagging-strand polymerase may arise from a protein-protein interaction that is established between a properly oriented and DnaB. Establishment of this contact literally cements the replication fork together, activating the helicase and ensuring rapid fork movement, as well as causing a rearrangement that makes beta refractory to the action of the -complex.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants GM36255 and GM34557 (to C. S. M. and K. J. M., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: kb, kilobase(s); Pol III HE, DNA polymerase III holoenzyme; TFII, tailed form II; SSB, E. coli single-stranded DNA-binding protein.

(^2)
Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J., Cell, in press.

(^3)
R.-D. Kim and C. S. McHenry, submitted for publication.

(^4)
M. Olson, J. Carter, H. G. Dallmann, and C. S. McHenry, manuscript in preparation.


REFERENCES

  1. Sakabe, K., and Okazaki, R. (1966) Biochim. Biophys. Acta 129, 652-654
  2. Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3511-3515 [Abstract]
  3. O'Donnell, M. (1987) J. Biol. Chem. 262, 16558-16565 [Abstract/Free Full Text]
  4. McHenry, C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748-1753 [Abstract]
  5. Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983 [Free Full Text]
  6. Kong, X. P., Onrust, R., O'Donnell, M., and Kuriyan, J. (1992) Cell 69, 425-432 [Medline] [Order article via Infotrieve]
  7. Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328-11334 [Abstract/Free Full Text]
  8. McHenry, C. S., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484 [Abstract]
  9. Wu, C. A., Zechner, E. L., and Marians, K. J. (1992) J. Biol. Chem. 267, 4030-4044 [Abstract/Free Full Text]
  10. Zechner, E. L., Wu, C. A., and Marians, K. J. (1992) J. Biol. Chem. 267, 4045-4053 [Abstract/Free Full Text]
  11. Zechner, E. L., Wu, C. A., and Marians, K. J. (1992) J. Biol. Chem. 267, 4054-4063 [Abstract/Free Full Text]
  12. Wu, C. A., Zechner, E. L., Hughes, A. J., Jr., Franden, M. A., McHenry, C. S., and Marians, K. J. (1992) J. Biol. Chem. 267, 4064-4073 [Abstract/Free Full Text]
  13. Wu, C. A., Zechner, E. L., Reems, J. A., McHenry, C. S., and Marians, K. J. (1992) J. Biol. Chem. 267, 4074-4083 [Abstract/Free Full Text]
  14. Hacker, K. J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 24221-24228 [Abstract/Free Full Text]
  15. Stukenberg, P. T., Turner, J., and O'Donnell, M. (1994) Cell 78, 877-887 [Medline] [Order article via Infotrieve]
  16. Mok, M., and Marians, K. J. (1987) J. Biol. Chem. 262, 16644-16654 [Abstract/Free Full Text]
  17. Model, P., and Zinder, N. (1974) J. Mol. Biol. 83, 231-251 [Medline] [Order article via Infotrieve]
  18. Minden, J., and Marians, K. J. (1985) J. Biol. Chem. 260, 9316-9325 [Abstract/Free Full Text]
  19. Marians, K. J. (1995) Methods Enzymol. 262, 507-521 [Medline] [Order article via Infotrieve]
  20. Johanson, K. O., Haynes, T. E., and McHenry, C. S. (1986) J. Biol. Chem. 261, 11460-11465 [Abstract/Free Full Text]
  21. Dallmann, H. G., Thimmig, R. L., and McHenry, C. S. (1995) J. Biol. Chem. 270, 29555-29562 [Abstract/Free Full Text]
  22. Olson, M. W., Dallmann, H. G., and McHenry, C. S. (1995) J. Biol. Chem. 270, 29570-29577 [Abstract/Free Full Text]
  23. Johanson, K. O., and McHenry, C. S. (1982) J. Biol Chem. 257, 12310-12315 [Abstract/Free Full Text]
  24. Engstrom, J., Wong, A., and Maurer, R. (1985) Genetics 113, 449-515
  25. McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663 [Abstract/Free Full Text]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.