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Curr.Opin.Chem. Biol., 15, 587-594 Available online at www.sciencedirect.com Bacterial replicases and related polymerases Charles S McHenry Bacterial replicases are complex, tripartite replicative replication that are conserved among all life forms are machines. They contain a polymerase, Pol III, a b2 processivity illustrated in Figure 1. factor and a DnaX complex ATPase that loads b2 onto DNA and chaperones Pol III onto the newly loaded b2. Many Structure and function of a, the catalytic bacteria encode both a full length t and a shorter g form of subunit DnaX by a variety of mechanisms. The polymerase catalytic Like all polymerases, Pol III a contains palm, thumb and subunit of Pol III, a, contains a PHP domain that not only binds fingers domains, in the shape of a cupped right hand 2+ to prototypical e Mg -dependent exonuclease, but also Figure 2. However, apo-enzyme structures of the full 2+ contains a second Zn -dependent proofreading length Thermus aquaticus (Taq) and a truncated version of exonuclease, at least in some bacteria. Replication of the E. coli (Eco) a subunit revealed a big surprise: the palm chromosomes of low GC Gram-positive bacteria require two domain has the basic fold of the X family of DNA Pol IIIs, one of which, DnaE, appears to extend RNA primers a polymerases, which includes the slow, non-processive only short distance before handing the product off to the major Pol bs [1 ,2 ]. replicase, PolC. Other bacteria encode a second Pol III (ImuC) that apparently replaces Pol V, required for induced A ternary complex of a dideoxy-terminated primer-tem- mutagenesis in E. coli. Approaches that permit simultaneous plate, incoming dNTP and full length Taq a provided biochemical screening of all components of complex bacterial significant insight into the function of Pol III a [3 ]. replicases promise inhibitors of specific protein targets and Among the template-primer induced conformational reaction stages. changes are movement of the thumb domain toward the DNA bound by the palm to make contacts with the sugar- Address phosphate backbone in the minor groove. The polymerase Department of Chemistry and Biochemistry, University of Colorado, contacts the template from its terminus to a position 12 Boulder 80309, United States nucleotides behind the primer terminus. The fingers domain creates a wall at the end of the primer terminus Corresponding author: McHenry, Charles S ([email protected]) that forces a sharp kink in the emerging template strand. In the ternary complex structure of a Gram-positive Pol Current Opinion in Chemical Biology 2011, 15:587–594 III, two novel elements, not present in Eco or Taq a, were 2+ This review comes from a themed issue on identified [4]. The four-Cys Zn binding motif, shown Molecular Machines earlier by Neal Brown and colleagues to be required for Edited by Stephen Benkovic and Kevin D. Raney activity, serves an apparently structural function and is not part of the catalytic site. DNA binding through the Available online 19th August 2011 thumb domain comes primarily from two b strands that 1367-5931/$ – see front matter interact with the minor groove [4]. # 2011 Elsevier Ltd. All rights reserved. PHP domain DOI 10.1016/j.cbpa.2011.07.018 A PHP domain (initially identified as having homology to phosphoesterases) is found in a wide variety of bacterial polymerases, including bacterial Pol bs (Figure 2). Initially, Introduction it was proposed that this region might be involved in Cellular chromosomal replicases from all branches of life pyrophosphate hydrolysis, but such an activity has not are tripartite. They contain a polymerase (Pol III in been found [1]. This domain, at least in Taq Pol III, 2+ bacteria and Pol d and e in eukaryotes), a sliding clamp contains a second proofreading activity that is Zn -de- 2+ processivity factor (b2 in bacteria and PCNA in eukar- pendent [5 ] and also binds the classical Mg -based proof- yotes), and a clamp loader (DnaX complex (DnaXcx) in reading subunit, e [6]. The structure of Taq a revealed a bacteria and RFC in eukaryotes). By themselves, repli- cluster of nine residues in the PHP domain that chelate cative polymerases do not exhibit special properties that three metal ions [2]. A structure of a Gram-positive PolC distinguish them from other polymerases, but together PHP domain also binds three metals, using the same nine with the sliding clamp and clamp loader they become ligands expected from the homologous Taq PHP structure. highly processive. b2 forms a ring that surrounds the Kuriyan and colleagues, from the structure of Eco a, DNA template and tethers the polymerase to it, enabling pointed out a channel between the polymerase active site processive replication. Other features of bacterial and the proposed PHP active site [1]. www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:587–594 588 Molecular Machines Figure 1 (a) (b) (c) 5´ 3´ β (E) 2 RNA (D) δ DnaG SSB ´ (A) DnaX Pol III Primer χψ γ δ Primase Complex Dna Xcx τ τ Primase (C) (B) DnaB SSB β β χ 3´ 2 2 Helicase ψ αεθ 5´ δ δ´ β Helicase α α γ τ τ ε ε Pol III αεθ θ θ β 5´ 3´ Current Opinion in Chemical Biology DNA Polymerase III Holoenzyme contacts at the replication fork. (a) A hexameric helicase translocates down the lagging strand template, splitting two strands apart in advance of the leading strand replicase, Pol III HE. Single-stranded regions of the lagging strand template are coated by SSB. Primase interacts with the helicase and synthesizes short RNA primers for Okazaki fragment synthesis that are extended by the Pol III HE until a signal is received to recycle to the next primer synthesized at the replication fork. For clarity, this view is drawn with a discrete DnaXcx on each Pol III; they are actually shared between the leading and lagging strand polymerase (dotted line). (b) Details of known subunit interactions within the Pol III HE. In addition, there is a transient interaction between d and, perhaps, additional DnaXcx subunits with b2 during the clamp loading reaction. (c) A cartoon of the replication fork showing relevant protein-protein interactions, including dimerization of the leading and lagging strand polymerases through contact of domain V of t with a. A contact between domain IV of two ts and two DnaB protomers anchors the replicase to the helicase, placing all replication fork components into one replisome. b2 binding domain the b1–b2 loop were changed to serine [8]. The proces- A structure of Taq a revealed a well-organized b2-binding sivity of the mutant polymerase was decreased, an effect domain with a HhH motif that together with its flanking that was rescued by the presence of the t-complex [8]. loops binds dsDNA [3] (Figure 2). The b2 binding con- The latter observation would seem to suggest that sensus sequence is presented in a loop that is oriented although the OB-fold contributes to ssDNA affinity adjacent to dsDNA as it exits the polymerase, in the and processivity, it is not the processivity sensor, or at correct position to bind b2 as it surrounds DNA. The b2 least that the residues mutated are not the key interactors. binding domain rotates 208 and swings down into position as the enzyme binds DNA [3], a reorientation that is In fact, the entire polymerase active site may function as apparently driven energetically by the HhH motif bind- the processivity switch [9 ]. Steitz and colleagues [3] have ing to DNA and probably coupled to conformational elegantly demonstrated a conformational change induced changes of the thumb, palm, OB fold, and PHP domains. by substrate binding in which several elements move that include placement of the b2 binding domain in a position OB-fold domain where it can productively interact with the b2 clamp on The structure of the ternary complex of Taq a with DNA. The geometry and spatial constraints around the primer-template and incoming dNTP reveals a striking active site when the exiting template is double-stranded conformational change that includes the OB-fold mov- might make insertion of the last nucleotide energetically ing to a position near the single-strand template distal unfavorable. Upon insertion, the product might lose to the primer [3]. The path of the emerging template, affinity for the active site, triggering a reversal of the which can be traced from electron density of the ribose- conformational changes that occurred upon primer-tem- phosphate backbone, appears to come close to the OB- plate and dNTP binding, switching the polymerase to a fold. The element of the OB-fold that comes closest to low-processivity mode. the ssDNA template, the b1–b2 loop, often contributes to ssDNA binding [7]. However, the b1–b2–b3 face t-binding domain that commonly interacts with ssDNA [7] appears to face The second half of the C-terminal domain in the Taq a away from the emerging template, toward the t-binding structure revealed a domain containing an incompletely domain. conserved sequence that binds weakly to b2, but is not required for processive replication in vitro or function in A test of the importance of the OB-fold motif was made vivo. This domain is loosely packed against the OB fold, using a mutant in which three basic residues located in with many polar residues in the interface [2] (Figure 2). Current Opinion in Chemical Biology 2011, 15:587–594 www.sciencedirect.com Bacterial replicases McHenry 589 Figure 2 (a) 778 511 964 272 1078 1160 430 560 D555 D401, 403 php palm thumb palm fingers β binding OB-fold τ binding β HhH binding loop (b) PHP Fingers Thumb Palm OB fold β-binding loop β binding τ binding Current Opinion in Chemical Biology Modular organization of Pol III a.
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