Molecular Microbiology (2003) 49 (5), 1157–1165 doi:10.1046/j.1365-2958.2003.03645.x© 2003 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 200349 511571165 Review Article C. S. McHenryChromosomal replicases as dimers Accepted 29 May, 2003. *For correspondence. [email protected]. MicroReview Chromosomal replicases as asymmetric dimers:studies of subunit arrangement and functional consequences Charles S. McHenry Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA. SummaryStudies of the DNA polymerase III holoenzyme of Escherichia coli support a model in which both theleading and lagging strand polymerases are heldtogether in a complex with the replicative helicaseand priming activities, allowing two identical aaaa cata-lytic subunits to assume different functions on thetwo strands of the replication fork. Creation of distinctfunctions for each of the two polymerases within theholoenzyme depends on the asymmetric character ofthe entire complex. The asymmetry of the holoenzymeis created by the DnaX complex, a heptamer thatincludes tttt and gggg products of the dnaX gene. tttt and gggg perform unique functions in the DnaX complex, andthe interaction between aaaa and tttt appears to dictate thecatalytic subunit’s role in the replicative reaction. Thisreview considers the properties of the DnaX complexincluding both tttt and gggg , with the goal of understandingthe properties of the replicase and its function in vivo .Recent studies in eukaryotic and other prokaryoticsystems suggest that an asymmetric dimeric repli-case may be universal. The leading and lagging strandpolymerases may be distinct in some systems. Forexample, Pol e and Pol d may function as distinctleading and lagging strand polymerases in eukary-otes, and PolC and DnaE may function as distinctleading and lagging strand polymerases in low GCcontent Gram-positive bacteria.General properties of replicases Cellular replicases from bacteria, archaea and eukaryotesare complex macromolecular assemblies that have ahighly conserved structure. The complexes are tripartiteassemblies that include a replicative polymerase, a ‘slidingclamp’ processivity factor ( b in bacteria and PCNA inarchaea and eukaryotes), and a complex multisubunitAT Pase (DnaX complex in bacteria and RFC in archaeaand eukaryotes) (see Fig. 1 for diagram of bacterial repli-case). The ATPase complex performs multiple essentialfunctions, one of which is to assemble the processivityfactor onto DNA, giving it the alternative name of ‘clamploader.’ b 2 and PCNA 3 are oligomeric ring-shaped mole-cules that encircle the DNA template and bind the poly-merase, forming a tether or clamp that holds thepolymerase on the DNA (Kuriyan and O’Donnell, 1993).Because a closed ring cannot assemble readily onto DNAby itself, it requires an ATP-powered machine to allow itto open and lock into place on the DNA. The ‘clamploaders’ form a pentameric ring composed of structurallysimilar proteins. Clamp loader subunits are members ofthe extended AAA + family of motor-like ATPases, that per-form a variety of cellular activities (Jeruzalmi et al .,2001a). In eukaryotes each of the five subunits areencoded by distinct genes.In bacteria, the five subunits are encoded by threegenes: dnaX , holA and holB . The DnaX subunit appearsto function as the sole ATPase in most bacterial DnaXcomplexes (Bullard et al ., 2002). In Escherichia coli andsome other bacteria, the dnaX gene encodes two prod-ucts, a shorter g subunit and a longer t protein. The g subunit is encoded by an alternative reading frame thatcan be created by several mechanisms including pro-grammed translational frameshifting in E. coli and tran-scriptional slippage in Thermus thermophilus (Blinkowaand Walker, 1990; Flower and McHenry, 1990; Tsuchi-hashi and Kornberg, 1990; Larsen et al ., 2000). Both g and t are ATPases and act as ‘clamp loading’ proteins.The longer protein t includes two domains not present in g ; these domains facilitate interaction between t and theDnaB helicase at the replication fork and between t andthe a subunit of Pol III (Dallmann et al ., 2000; Gao andMcHenry 2001a, b). The E. coli DnaX complex alsoincludes one copy each of the c and y subunits. y bindsto DnaX and c , tethering the latter protein to the complex.1158 C. S. McHenry © 2003 Blackwell Publishing Ltd, Molecular Microbiology , 49 , 1157–1165 c interacts with single-stranded DNA binding protein(SSB) with a much higher affinity when SSB is bound toDNA than when it is free in solution (Glover and McHenry,1998). Because DNA replication models include single-stranded DNA only on the lagging strand of the replicationfork, it has been suggested that cy is associated withlagging strand replication (Kelman et al ., 1998) (Fig. 1,right). The c - y -SSB contact is essential for robust repli-case activity in solutions at physiological ionic strength(Glover and McHenry, 1998).Assembly of the replicative complex is often describedas involving two stages, although this distinction is some-what artificial. In the absence of polymerase, the DnaXcomplex can transfer b onto DNA in a reaction thatrequires ATP hydrolysis. Pol III can associate with theloaded b 2 and form a highly processive complex. How-ever, in vivo , the DnaX complex is likely to contain t and g , so that it also can bind to a . Under these conditions,the functional replicase would assemble onto DNA intightly coupled, nearly concerted steps. The distinctionbetween the ‘concerted’ reaction and the two-stage reac-tion is revealed when assembly is compared in the pres-ence of ATP or the non-hydrolysable analogue of ATP,AT P g S. In the presence of ATP g S, the initiation complexforms only if t is present in the DnaX complex, revealingan advantage of a being held in a complex with the‘clamp loader’ during the b ‘loading’ reaction (Dallmann et al ., 1995). In addition, in the presence of ATP g S, theamount of complex formed is reduced by half. Further,when ATP g S is added to purified initiation complexesformed in the presence of ATP, half of the initiationcomplexes dissociate (Johanson and McHenry, 1984;McHenry and Johanson, 1984; Glover and McHenry,2001).Previous studies, including the