Molecular Cell, Vol. 4, 541–553, October, 1999, Copyright 1999 by Cell Press

Replisome Assembly at oriC, the Replication Origin of E. coli, Reveals an Explanation for Initiation Sites outside an Origin

Linhua Fang,*^§ Megan J. Davey,^† and Mike O’Donnell^†‡

*Microbiology Department Joan and Sanford I. Weill Graduate School of Medical Sciences of Cornell University New York, New York 10021 ^† The Rockefeller University and have not been addressed. For example, is the local un- winding sufficiently large for two helicases to assemble for bidirectional replication, or does one helicase need to enter first and expand the bubble via helicase action to make room for the second helicase? The known rep- licative helicases are hexameric and encircle ssDNA. Which strand does the initial helicase(s) at the origin encircle, and if there are two, how are they positioned relative to one another? Primases generally require at least transient interaction with helicase to function. Can primase function with the helicase(s) directly after heli- case assembly at the origin, or must helicase-catalyzed DNA unwinding occur prior to RNA primer synthesis? Chromosomal replicases are comprised of a ring-shaped protein clamp that encircles DNA, a clamp-loading com- plex that uses ATP to assemble the clamp around DNA, and a DNA polymerase that binds the circular clamp, thereby remaining tightly bound to DNA for highly pro- cessive synthesis (Kelman and O’Donnell, 1995). Can this entire replicating machinery assemble at the origin with the helicase before DNA unwinding, or does this assembly process occur in a separate stage after the helicases generate sufficient space to accommodate these large machines? If so, how much space is needed? This report addresses these questions through a study of the stepwise events leading to the point at which two replisomes are assembled in opposite direc- tions at the E. coli origin, oriC. The study utilizes 19 different proteins produced by recombinant methods. The E. coli chromosome is replicated bidirectionally from oriC (Kornberg and Baker, 1992). Several copies of the initiator protein, DnaA, bind specifically to at least four 9-mer DNA-binding sites within the 245 bp oriC (Fuller et al., 1984). In the presence of ATP, the interac- tion of DnaA with oriC melts 26 bp within a region of three tandem 13-mer AT-rich repeats at one end of oriC to form the “open complex” (Bramhill and Kornberg, 1988; Gille and Messer, 1991). Open complex formation is aided by HU protein or IHF (integration host factor) (Hwang and Kornberg, 1992). Following this, at least one molecule of the replicative helicase, the hexameric DnaB protein, assembles onto the origin in a reaction that depends on DnaC protein to form the prepriming com- plex (Baker et al., 1987; Funnell et al., 1987; Bramhill and Kornberg, 1988). Electron microscopy studies suggest that DnaB binds oriC in the vicinity of the open complex (Funnell et al., 1987). ATP is needed by DnaA and DnaC in these initial steps, and therefore, the DnaB helicase is active and mobile during this assembly reaction. The mobility of DnaB has prevented determination of its ex- act assembly site(s) at oriC and whether one or two DnaB hexamers assemble at oriC before helicase action. Steps downstream of DnaB assembly have not been described in detail but must include priming by primase (DnaG), and the assembly of two molecules of the chro- mosomal replicase, DNA polymerase III holoenzyme, to form bidirectional replication forks.
Howard Hughes Medical Institute New York, New York 10021

Summary This study outlines the events downstream of origin unwinding by DnaA, leading to assembly of two repli- cation forks at the E. coli origin, oriC. We show that two hexamers of DnaB assemble onto the opposing strands of the resulting bubble, expanding it further, yet helicase action is not required. Primase cannot act until the helicases move 65 nucleotides or more. Once primers are formed, two molecules of the large DNA polymerase III holoenzyme machinery assemble into the bubble, forming two replication forks. Primer loca- tions are heterogeneous; some are even outside oriC. This observation generalizes to many systems, pro- karyotic and eukaryotic. Heterogeneous initiation sites are likely explained by primase functioning with a mov- ing helicase target.

Introduction

Replication origins are cis-acting sequences that direct the assembly of proteins onto DNA for the replication process, producing two copies of the genetic material (Kornberg and Baker, 1992). Generally, initiation from an origin requires an origin-binding protein, which may act in concert with other DNA-binding factors to locally unwind an AT-rich region within the origin sequence. This unwound bubble region is thought to provide an entry point leading to assembly of two helicases that move in opposite directions. Two copies of the primase and replicase must also be recruited to form two replica- tion forks for bidirectional synthesis of the DNA. Events leading to localized unwinding of DNA within the origin have been characterized in several systems, including the E. coli origin, oriC (Bramhill and Kornberg, 1988); the bacteriophage ␭ origin, ori ␭ (Schnos et al., 1988); the origins of E. coli plasmids R6K (Mukherjee et al., 1985) and RK2 (Konieczny et al., 1997); bacterio- phage P1 (Mukhopadhyay and Chattoraj, 1993); and ori- gins of viruses SV40 (Borowiec et al., 1990), HSV-1 (Lee and Lehman, 1997), EBV (Frappier and O’Donnell, 1992), and BPV (Gillette et al., 1994). However, important ques- tions about events that occur downstream of this step

‡

To whom correspondence should be addressed (e-mail: odonnel@ rockvax.rockefeller.edu).

§

This report examines origin activation events that oc- cur downstream of the DnaA/HU-mediated open com-

Present address: Molecular Staging Inc., 66 High Street, Guilford, Connecticut 06437.
Molecular Cell 542

plex. The position and stoichiometry of DnaB at oriC has been determined in advance of helicase-catalyzed unwinding using a mutant of DnaB that assembles onto DNA but is inactive as a helicase. The results demon- strate that DnaC delivers two DnaB hexamers to oriC in the absence of helicase action; DnaC does not remain on the DNA. DnaB assembly onto oriC results in an expansion of the ssDNA in the open complex unaided by helicase activity. Footprint analysis indicates that the two helicases are positioned face to face, one on each strand, such that they pass each other early in the un- winding process. Primase is unable to prime oriC in the absence of helicase action, even though the origin contains two DnaB hexamers. For want of a primed site, replication forks containing DNA polymerase III holoen- zyme also do not assemble in the absence of helicase action. Hence, origin activation proceeds to the point of assembly of two helicases onto DNA but thereafter requires helicase motion for priming and assembly of replication forks. of the origin. In the absence of gyrase, the unwinding of a supercoiled 6.6 kb plasmid by DnaB helicase is restricted to approximately 6.4% Ϯ 2.4% of the plasmid length, being limited by the topological tension gener- ated upon unwinding a closed duplex circle (Baker et al., 1987). The oriC sequence was cloned into pUC18 to generate pUC18oriC (3.1 kb final), and the extent of unwinding by DnaB in the absence of gyrase was examined by measuring the length of nascent leading strand chains (as in Hiasa et al., 1996). The largest length of these nascent chains averaged 65–83 nucleotides, which indicates a bubble size of 66–99 nucleotides, as- suming the start sites mapped later in this study, in Figure 5. This value is within 2-fold of the 6.4% deter- mined by the earlier electron microscopy study. To de- termine whether two DnaB hexamers, and one or two molecules of DNA polymerase III holoenzyme, could as- semble onto this unwound DNA, we radiolabeled pro- teins and added them to either pUC18oriC or pUC18. The stoichiometry of proteins bound to the DNA was then determined by gel filtration analysis using the large pore BioGel A-15m resin. The large plasmid DNA and proteins bound to it elute early in the excluded volume (fractions 10–15); proteins not bound to DNA are in- cluded and elute later (fractions 18–35). Five experi- ments were performed using DnaA, HU, DnaB, DnaC, DnaG, SSB, Pol III* (DNA polymerase III holoenzyme lacking only ␤), and ␤ in which either ³²P-DnaB, ³H-DnaC,
Helicase-mediated unwinding of approximately 100 nucleotides is sufficient for primase action on both strands, and for assembly of two molecules of the DNA polymerase III holoenzyme machinery. We propose that each DNA polymerase III holoenzyme extends these ini- tial two primers across the replication bubble until they encounter, and couple, with the DnaB on the opposite strand.

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The RNA/DNA junctions of the primed start sites in this in vitro system demonstrate that they are dispersed, occurring at any of several positions, and are located inside and outside the origin. Heterogeneous location of primed sites is consistent with earlier in vivo mapping studies (Hirose et al., 1983; Kohara et al., 1985). The nearest primed sites to the initial position of DnaB at oriC imply that each DnaB moves 65 nucleotides or more before primase synthesizes an RNA primer. The requirement of DnaB to move for primase to act on oriC provides an explanation for the heterogeneity of initiating RNA/DNA junctions. Namely, since primase re- quires at least transient interaction with DnaB to func- tion, it must associate with a moving target, resulting in dispersal of primed sites in the vicinity of the origin. This finding may generalize, as heterogeneous location of start sites is observed in several replication systems, including bacteriophage ␭, (Tsurimoto and Matsubara, 1984; Yoda et al., 1988); R6K plasmid (Chen et al., 1998); plasmid R1 (Bernander et al., 1992); the simian virus, SV40 (Hay and DePamphilis, 1982; Bullock and Denis, 1995); and the S. cerevisiae origin, ARS1 (Bielinsky and Gerbi, 1998). Based on these studies of E. coli oriC, it seems likely that heterogeneity in priming sites at origins of other systems will also be explained by the need for helicase motion to support primase action.
³²P-primase, H-Pol III*, or H-␤ was substituted for its unlabeled counterpart. The results, in Figure 1 (quantitated in Table 1), indi- cate that approximately two molecules of DnaB (as hexamer), two molecules of Pol III*, and two ␤ clamps assemble onto pUC18oriC. Primase did not bind pU- C18oriC, and less than one DnaC monomer was re- tained, consistent with the known distributive action of primase (Marians, 1992) and apparent absence of DnaC in the prepriming complex at oriC (Funnell et al., 1987). Interaction of DnaB, Pol III*, and ␤ with DNA required the oriC sequence as illustrated in Figure 1 for the plasmid lacking oriC (pUC18). Presumably, two replisomes, each containing one DnaB hexamer, one Pol III* (each having two core DNA polymerases), and one ␤ clamp, assem- bled onto each molecule of pUC18oriC (recovery of DNA was typically 85%–95%). These isolated protein–DNA complexes are func- tional, requiring only gyrase for activity. Further, the iso- lated complexes display bidirectional replication from oriC (not shown, but experimental design and results are similar to those of Figure 5D). It is interesting to note that only one ␤ clamp is present for the two core polymerases within Pol III*. Thus, only the leading (or lagging) strand core polymerase is tethered to DNA by a ␤ clamp. It is unlikely that one replisome contains two ␤ clamps, one for each core polymerase, and the other replisome lacks ␤, as additional studies not shown here did not detect Pol III* on pUC18oriC in reactions lack- ing ␤.

Results A Small Bubble at oriC Supports Assembly of Two Replisomes

In this first experiment, we asked whether two full repli- somes can assemble onto DNA upon constraining the unwinding of the plasmid to a small area in the vicinity

How Far Does Orisome Assembly Proceed in the Absence of Helicase Action?

The experiment of Figure 1 demonstrated that two repli- cation forks assemble on an oriC plasmid in the absence

Replisome Assembly at oriC 543

The ssDNA created by binding of the DnaA/HU to form open complex is approximately 26 nucleotides of melted DNA, as determined by KMnO₄ modification (Gille and Messer, 1991). Is this DNA structure sufficient to support assembly of two helicases, two priming events, and two molecules of DNA polymerase III holoenzyme in the ab- sence of further unwinding catalyzed by DnaB? The DnaB hexamer forms a ring (San Martin et al., 1995; Yu et al., 1996) that encircles ssDNA (Yuzhakov et al., 1996) and appears to require 20 Ϯ 3 nucleotides of ssDNA for tight binding (Jezewska et al., 1996). From this informa- tion, a 26-residue “bubble” would accommodate only one molecule of DnaB, although it may be possible that each strand of the bubble could accommodate one DnaB hexamer. Our strategy to determine whether one or two DnaB hexamers, and other components of the replisome, as- semble into the open complex at oriC without helicase action was to construct a DnaB mutant that lacks heli- case activity. The sequence of DnaB contains a Walker A nucleotide-binding motif between amino acid residues 223 and 244. We replaced Lys-236, the most conserved residue in the Walker A motif, with either Arg or Ala. The mutations were introduced into a dnaB gene that also includes a cAMP-dependent protein kinase motif in the N terminus (Yuzhakov et al., 1996). The N-terminal modi- fication, which has little effect on the activities of DnaB (Yuzhakov et al., 1996), enables radiolabeling of the pro- tein using [␥-³²P]ATP and a protein kinase. In addition to supporting in vitro oriC replication, wild- type DnaB has many other enzymatic activities. These DnaB activities include: ATP binding, ssDNA-dependent ATP hydrolysis, duplex DNA unwinding, and activation of primase on an ssDNA template (general priming activ- ity). In Figure 2, we characterized the ATP site mutants of DnaB by comparing these enzymatic activities with those of wild-type DnaB. The results show that substitution of Arg for Lys-236
(DnaB^PKK236R) resulted in a DnaB that retained full ac- tivity in all four assays tested (Figures 2A–2E). As we desire a DnaB lacking helicase activity, the K236R mu- tant is not discussed further in this report. Replacement of Lys-236 with Ala (DnaB^PKK236A) resulted in the de- sired DnaB mutant; it had only weak ATPase activity (Figure 2B), no detectable helicase activity (Figure 2C), and no activity in the oriC replication system (Figure 2E). However, DnaB^PKK236A retained the ability to bind ATP (K_d ϭ 7 ␮M, Figure 2A), and to support primase action in general priming (Figure 2D) (DnaB^PKK236A is still a hexamer, not shown). In fact, DnaB^PKK236A was about 3-fold more active than DnaB^PK in general priming, con- sistent with an earlier study of a DnaB mutant defective in ATP hydrolysis (Shrimankar et al., 1992).

Figure 1. Assembly of Proteins at the Origin Five different experiments, differing only in which radioactive-labeled protein was added in place of the unlabeled protein, were performed as described in Experimental Procedures. Proteins were assembled onto plasmid DNA either containing oriC (closed circles), or lacking it (open circles) followed by gel filtration. Radiolabeled proteins were located in column fractions by liquid scintillation and quantitated from their known specific activity: (A) ³²P-DnaB, (B) ³H-DnaC, (C) ³H-Pol III*, (D) ³H-␤, and (E) ³²P-primase. The diagram at the top summarizes the results. Two hexamers of DnaB are shown as encir- cling ssDNA. The two ring-shaped ␤ clamps are each shown as a torus encircling a primed site. Each Pol III* contains two core polymerases and one ␥ complex clamp loader (not shown).