Replisome Assembly at Oric, the Replication Origin of E. Coli, Reveals an Explanation for Initiation Sites Outside an Origin

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,² and Mike O'Donnell²³ have not been addressed. For example, is the local un- *Microbiology Department winding sufficiently large for two helicases to assemble Joan and Sanford I. Weill Graduate School of Medical for bidirectional replication, or does one helicase need Sciences of Cornell University to enter first and expand the bubble via helicase action New York, New York 10021 to make room for the second helicase? The known rep- ² The Rockefeller University and licative helicases are hexameric and encircle ssDNA. Howard Hughes Medical Institute Which strand does the initial helicase(s) at the origin New York, New York 10021 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 Summary primase function with the helicase(s) directly after helicase assembly at the origin, or must helicase-catalyzed This study outlines the events downstream of origin DNA unwinding occur prior to RNA primer synthesis? unwinding by DnaA, leading to assembly of two repli- Chromosomal replicases are comprised of a ring-shaped cation forks at the E. coli origin, oriC. We show that protein clamp that encircles DNA, a clamp-loading com- two hexamers of DnaB assemble onto the opposing plex that uses ATP to assemble the clamp around DNA, strands of the resulting bubble, expanding it further, and a DNA polymerase that binds the circular clamp, yet helicase action is not required. Primase cannot act thereby remaining tightly bound to DNA for highly pro- until the helicases move 65 nucleotides or more. Once cessive synthesis (Kelman and O'Donnell, 1995). Can primers are formed, two molecules of the large DNA this entire replicating machinery assemble at the origin polymerase III holoenzyme machinery assemble into with the helicase before DNA unwinding, or does this the bubble, forming two replication forks. Primer loca- assembly process occur in a separate stage after the tions are heterogeneous; some are even outside oriC. helicases generate sufficient space to accommodate This observation generalizes to many systems, pro- these large machines? If so, how much space is needed? karyotic and eukaryotic. Heterogeneous initiation sites This report addresses these questions through a are likely explained by primase functioning with a mov- study of the stepwise events leading to the point at ing helicase target. which two replisomes are assembled in opposite directions at the E. coli origin, oriC. The study utilizes 19 different proteins produced by recombinant methods. Introduction The E. coli chromosome is replicated bidirectionally from oriC (Kornberg and Baker, 1992). Several copies Replication origins are cis-acting sequences that direct of the initiator protein, DnaA, bind specifically to at least the assembly of proteins onto DNA for the replication four 9-mer DNA-binding sites within the 245 bp oriC process, producing two copies of the genetic material (Fuller et al., 1984). In the presence of ATP, the interac- (Kornberg and Baker, 1992). Generally, initiation from tion of DnaA with oriC melts 26 bp within a region of an origin requires an origin-binding protein, which may three tandem 13-mer AT-rich repeats at one end of oriC act in concert with other DNA-binding factors to locally to form the ªopen complexº (Bramhill and Kornberg, unwind an AT-rich region within the origin sequence. 1988; Gille and Messer, 1991). Open complex formation This unwound bubble region is thought to provide an is aided by HU protein or IHF (integration host factor) entry point leading to assembly of two helicases that (Hwang and Kornberg, 1992). Following this, at least one move in opposite directions. Two copies of the primase molecule of the replicative helicase, the hexameric DnaB and replicase must also be recruited to form two replica- protein, assembles onto the origin in a reaction that tion forks for bidirectional synthesis of the DNA. depends on DnaC protein to form the prepriming com- Events leading to localized unwinding of DNA within plex (Baker et al., 1987; Funnell et al., 1987; Bramhill and the origin have been characterized in several systems, Kornberg, 1988). Electron microscopy studies suggest including the E. coli origin, oriC (Bramhill and Kornberg, that DnaB binds oriC in the vicinity of the open complex 1988); the bacteriophage l origin, ori l (Schnos et al., (Funnell et al., 1987). ATP is needed by DnaA and DnaC 1988); the origins of E. coli plasmids R6K (Mukherjee et in these initial steps, and therefore, the DnaB helicase al., 1985) and RK2 (Konieczny et al., 1997); bacterio- is active and mobile during this assembly reaction. The phage P1 (Mukhopadhyay and Chattoraj, 1993); and ori- mobility of DnaB has prevented determination of its ex- gins of viruses SV40 (Borowiec et al., 1990), HSV-1 (Lee act assembly site(s) at oriC and whether one or two and Lehman, 1997), EBV (Frappier and O'Donnell, 1992), DnaB hexamers assemble at oriC before helicase action. and BPV (Gillette et al., 1994). However, important ques- Steps downstream of DnaB assembly have not been tions about events that occur downstream of this step described in detail but must include priming by primase (DnaG), and the assembly of two molecules of the chromosomal replicase, DNA polymerase III holoenzyme, to ³ To whom correspondence should be addressed (e-mail: odonnel@ rockvax.rockefeller.edu). form bidirectional replication forks. § Present address: Molecular Staging Inc., 66 High Street, Guilford, This report examines origin activation events that oc- Connecticut 06437. cur downstream of the DnaA/HU-mediated open com- Molecular Cell 542 plex. The position and stoichiometry of DnaB at oriC of the origin. In the absence of gyrase, the unwinding has been determined in advance of helicase-catalyzed of a supercoiled 6.6 kb plasmid by DnaB helicase is unwinding using a mutant of DnaB that assembles onto restricted to approximately 6.4% 6 2.4% of the plasmid DNA but is inactive as a helicase. The results demon- length, being limited by the topological tension gener- strate that DnaC delivers two DnaB hexamers to oriC ated upon unwinding a closed duplex circle (Baker et in the absence of helicase action; DnaC does not remain al., 1987). The oriC sequence was cloned into pUC18 on the DNA. DnaB assembly onto oriC results in an to generate pUC18oriC (3.1 kb final), and the extent expansion of the ssDNA in the open complex unaided of unwinding by DnaB in the absence of gyrase was by helicase activity. Footprint analysis indicates that the examined by measuring the length of nascent leading two helicases are positioned face to face, one on each strand chains (as in Hiasa et al., 1996). The largest length strand, such that they pass each other early in the un- of these nascent chains averaged 65±83 nucleotides, winding process. Primase is unable to prime oriC in which indicates a bubble size of 66±99 nucleotides, as- the absence of helicase action, even though the origin suming the start sites mapped later in this study, in contains two DnaB hexamers. For want of a primed site, Figure 5. This value is within 2-fold of the 6.4% deter- replication forks containing DNA polymerase III holoen- mined by the earlier electron microscopy study. To de- zyme also do not assemble in the absence of helicase termine whether two DnaB hexamers, and one or two action. Hence, origin activation proceeds to the point molecules of DNA polymerase III holoenzyme, could as- of assembly of two helicases onto DNA but thereafter semble onto this unwound DNA, we radiolabeled pro- requires helicase motion for priming and assembly of teins and added them to either pUC18oriC or pUC18. replication forks. The stoichiometry of proteins bound to the DNA was Helicase-mediated unwinding of approximately 100 then determined by gel filtration analysis using the large nucleotides is sufficient for primase action on both pore BioGel A-15m resin. The large plasmid DNA and strands, and for assembly of two molecules of the DNA proteins bound to it elute early in the excluded volume polymerase III holoenzyme machinery. We propose that (fractions 10±15); proteins not bound to DNA are in- each DNA polymerase III holoenzyme extends these ini- cluded and elute later (fractions 18±35). Five experi- tial two primers across the replication bubble until they ments were performed using DnaA, HU, DnaB, DnaC, encounter, and couple, with the DnaB on the opposite DnaG, SSB, Pol III* (DNA polymerase III holoenzyme strand. lacking only b), and b in which either 32P-DnaB, 3H-DnaC, The RNA/DNA junctions of the primed start sites in 32P-primase, 3H-Pol III*, or 3H-b was substituted for its this in vitro system demonstrate that they are dispersed, unlabeled counterpart. occurring at any of several positions, and are located The results, in Figure 1 (quantitated in Table 1), indi- inside and outside the origin. Heterogeneous location cate that approximately two molecules of DnaB (as of primed sites is consistent with earlier in vivo mapping hexamer), two molecules of Pol III*, and two b clamps studies (Hirose et al., 1983; Kohara et al., 1985). The assemble onto pUC18oriC. Primase did not bind pU- nearest primed sites to the initial position of DnaB at C18oriC, and less than one DnaC monomer was re- oriC imply that each DnaB moves 65 nucleotides or tained, consistent with the known distributive action of more before primase synthesizes an RNA primer. The primase (Marians, 1992) and apparent absence of DnaC requirement of DnaB to move for primase to act on in the prepriming complex at oriC (Funnell et al., 1987).

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