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Protein-Protein Interactions in the Bacterial Replisome

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Protein-Protein Interactions in the Bacterial Replisome SHOWCASE ON RESEARCH Protein-Protein Interactions in the Bacterial Replisome Patrick Schaeffer, Madeleine Headlam and Nick Dixon Research School of Chemistry, Australian National University, ACT 0020 Replication of DNA in all organisms proceeds in proteins, while others like the small single-stranded three stages: initiation at origins of replication, DNA (SS) DNA phages make use of subsets of host synthesis (elongation) at replication forks, and enzymes. Similar reductionist approaches have more termination. Each of these processes is mediated by recently been applied to identify many of the multiple stable or transient protein-protein and replication proteins from eukaryotes. It is clear that protein-DNA interactions involving subsets of the 30 protein functions and mechanisms are conserved in or so different replication proteins (Fig. 1). The all organisms (Table 1), though the proteins may elongation phase occurs within a large nucleoprotein have little or no recognisable sequence similarity. assembly called the replisome (1). Replisomes have Work in the last ten years has produced a good n e v e r b e e n i s o l a t e d i n t a c t f r o m c e l l s , s o a n understanding of how protein-protein interactions understanding of their structure and function has mediate replisomal DNA synthesis (Fig. 2A), and required their painstaking in vitro reassembly from high-resolution structures of many of the individual individual subunits. Mechanistic studies have made proteins (or domains thereof) and some larger extensive use of the Escherichia coli proteins, in part complexes are now available. For all of these reasons, because they are separately isolatable in large it is apparent that E. coli DNA replication provides quantities from overproducing strains, and in vitro an excellent opportunity to study the chemistry that initiation, elongation and termination reactions have governs macromolecular interactions in this complex, been faithfully reconstituted using defined DNA flexible and dynamic nucleoprotein assembly. The E. templates. Other systems studied in similar detail coli replisome can thus be seen as a tractable model include the replication of the DNA of bacteriophages. system to develop knowledge and tools that may be Some, like T4 and T7, encode their own replication applied to other large dynamic multiprotein complexes. Such complexes mediate or DnaA/dnaA box control many aspects of development and HU FIS function in organisms from all domains of life. INITIATOR Assembly of two replisomes at oriC IHF How replisomes are assembled is probably the least well-understood process in P R IMAS E HE LIC AS E LOADE R replication. In the 4.7 million base pairs of the circular E. coli chromosome is a 260-base DnaG DnaC pair sequence that constitutes the unique origin of replication (oriC). The origin is recognised by the DnaA replication initiator protein (2, 3). Multiple copies of DnaA bind DnaB HE LIC AS E to five 'DnaA boxes' in oriC, and in the presence of small basic histone-like proteins (HU and/or IHF) that stabilise bends in S S B Tus/Ter DNA, this leads to separation of the two DNA strands at a nearby AT-rich region to TE R MINATOR produce a single-stranded bubble. The ring-shaped hexameric DnaB6 helicase χ γ τ is a molecular motor that hydrolyses ATP to fuel its translocation in the 5′-3′ direction on ψ δ' τ α ε θ t h e s t r a n d i t e n c i r c l e s , r e s u l t i n g i n δ displacement of the other strand at a replication fork (4, 5). When it is free in the C LAMP LOADE R P OLYME R AS E cell, DnaB6 is normally associated with six molecules of the helicase loader protein D n a C . T h e c e n t r a l c h a n n e l i n t h e DnaB6.(DnaC)6 complex is closed (6) so that β β C LAMP the helicase cannot associate with DNA until it interacts specifically with the N- Fig. 1. Schematic representation of the E. coli replicative terminal domain of DnaA in the origin interactome. complex. This leads to opening of the Vol 35 No 1 April 2004 AUSTRALIAN BIOCHEMIST Page 9 SHOWCASE ON Interactions in the Replisome RESEARCH Table 1. Correspondence among replication proteins from Escherichia coli, Bacillus subtilis, and bacteriophages T7 and T4. Function E. coli B. subtilis Phage T7 Phage T4 Polymerase/exonuclease αεθ PolC, DnaEBS Gp5 Gp43 Sliding clamp β2 β2 Trx (E. coli) Gp453 Clamp loader δγτ2δ′ψχ τ5 − Gp444Gp62 Terminator Tus RTP2 − − SSB SSB4 SSB4 Gp2.52 Gp32 Initiator DnaA, IHF, DnaA, B, D T7 RNAP − HU, FIS Replicative helicase DnaB6 DnaC6 Gp4 Gp41 Helicase loader DnaC DnaI − Gp59 Primase DnaG DnaG Gp4 Gp61 helicase ring, dissociation of DnaC, and directional smaller C-terminal region that associates tightly with α. loading of two DnaB6 molecules within the SS-DNA The β 2 molecule is a ring-shaped dimer that regions. The helicase molecules extend the single- encircles double-stranded DNA without actually stranded bubble by translocating in opposite contacting it to form a tight topological linkage (10- directions on what will become the lagging strands 12). It acts as a sliding clamp on the newly-replicated of the two replication forks that invade the duplex leading and lagging strands behind the two Pol III DNA in opposite directions on each side of the cores, with which it interacts through a C-terminal origin. Each molecule of the helicase transiently region of the α subunit. This ensures that the associates through its N-terminal region with the C- polymerase never dissociates at an inappropriate terminal domain of the DnaG primase. Primase is a time from the template-primer. The third sub- specialist RNA polymerase that periodically assembly (δ.γ.τ2.δ′)-(ψ.χ) is the clamp loader or γ synthesises short RNA primers on the SS-DNA complex, which has multiple functions (7, 11). The τ r e g i o n s t o i n i t i a t e D N A s y n t h e s i s b y D N A and γ subunits are closely related molecules. They polymerase III (Pol III). DNA polymerases cannot are both translated from the same (dnaX) mRNA. start polynucleotide chains, so each nascent DNA About half of the time, the ribosome undergoes a fragment must be primed by DnaG. On the leading programmed frameshift and terminates translation strand, the first primer laid down on each strand at two-thirds of the way through the message to oriC is extended continuously and processively by produce γ. Thus, the N-terminal regions of γ and τ Pol III, which may never dissociate from the are identical, and it is these regions that associate template. On the other hand, replication of the together and with δ.δ′ in the central five-subunit core lagging strand needs to occur discontinuously, of the clamp loading complex (7, 13). The distinct C- producing Okazaki fragments about 1 kb in length terminal regions of the two molecules of τ have two that are subsequently processed by DNA polymerase functions. One is to dimerise the core, through I and joined by DNA ligase. Thus, the lagging-strand interaction with the C-terminal regions of the two α Pol III must dissociate and reassociate with a new subunits. The other is to interact with the DnaB6 primer every second or two. molecule on the lagging strand, ensuring continued communication between the holoenzyme and the Elongation by DNA polymerase III holoenzyme helicase (7). The Pol III holoenzyme (Fig. 2B) is remarkable not The third function of the γ complex is to act as the only because it is a very efficient enzyme (turnover clamp loader (11). The δ subunit interacts with the β2 number about 1000 s-1 per active site), but also sliding clamp, probably at the site occupied by α (see because it is almost perfectly faithful (it only the following articles in this Showcase on Research incorporates correctly paired nucleotides) and on β2 by Wijffels et al. and its eukaryotic equivalent infinitely processive (never dissociates from a PCNA, by Kunz et al.). On the lagging strand, in a template-primer). These attributes require the series of conformational changes in the γ complex collaboration of 10 different subunits; the probable triggered by the arrival of a new primer terminus c o m p o s i t i o n o f t h e h o l o e n z y m e i s ( α . ε . θ ) 2 - and driven by hydrolysis of ATP, δ acts as a wrench (δ.γ.τ2.δ′.ψ.χ)−(β2)2, comprising three separate sub- to open the β2 ring and load it onto the template- assemblies (7). The core complex (α.ε.θ) contains the primer (14, 15). It is not clear yet if the same large polymerase subunit (α), the proofreading 3′-5′ mechanism is used at oriC to load the β2 molecules exonuclese (ε) that ensures high fidelity by excising that continuously associate with the leading strand nucleotides misincorporated by the polymerase (8), polymerase. Indications that β2 interacts with DnaA and the small θ subunit that binds to and stabilises ε (and perhaps other components of the initiatior (9). The ε subunit has two domains, a larger N- complex) suggest that they might be loaded in a terminal portion that has enzymatic activity, and a different way.
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