Although the two alternative Escherichia coli dnaX gene products, and ␥, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing but not ␥. We developed a new assay based on the binding of complexes containing biotin-tagged to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III

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... subunits on the kinetics of co-assembly of ␥ and into the same complex. Auxiliary proteins in combination with ␦ almost completely blocked co-assembly, whereas or ␦ alone slowed the association only moderately compared with the interaction of with ␥ alone. In contrast, DNA polymerase III core, in the absence of ␦␦ and , accelerated the coassembly of and ␥, suggesting a role for DNA polymerase III [ 2 (pol III core) 2 ] in the assembly pathway of holoenzyme. The Escherichia coli chromosome is replicated by the DNA polymerase III holoenzyme, 1 which contains three functional subassemblies: pol III core, the ␤ sliding clamp processivity factor, and the DnaX complex. The pol III core contains the ␣, ⑀, and subunits and provides the polymerase function. The multiprotein DnaX complex recognizes the primer terminus, loads ␤ onto DNA in an ATP-dependent manner, and functions as a communications and organizational node for the various replication and primosomal proteins at the replication fork (1-9). The DnaX complex contains the ATPases and ␥, which are the alternative frameshift products of the dnaX gene, plus the auxiliary subunits ␦, ␦Ј, , and . The subunit, but not the shorter translation product ␥, dimerizes the pol III core through interactions between structural domain V of and the ␣ subunit to coordinate leading and lagging strand synthesis (6, 10 -12). There is also a -mediated interaction between holoenzyme and DnaB that is essential for coupling the replicase and the primosomal apparatus at the replication fork (4, 7, 9, 12) . In the elongation complex, protects ␤ 2 from removal by exogenous ␥ complex, increasing the processivity of the repli-case (12). The subunit also is a bridge between ␣ and a single-stranded DNA-binding protein interaction, strengthening the holoenzyme interactions with the protein that coats the lagging strand at the replication fork (13, 14) . Within the DnaX complex, ␦Ј and bind directly to ␥; ␦ binds ␦Ј, and binds (5, 15 ). The DnaX-␦Ј and DnaX-interactions occur through structural domain III, which is common to both and ␥ (8). It is also known that ␦ and ␦Ј form a 1:1 complex and together with DnaX load ␤ onto primed templates. (16, 17) . The and subunits also form a 1:1 complex and increase the affinity of DnaX for ␦ and ␦Ј such that a functional DnaX complex can be assembled at physiological subunit concentrations (8, 13, 15, 18) . Various forms of the clamp-loading complex have been characterized including a ␥ complex (␥ 3 ␦␦Ј), a complex ( 3 ␦␦Ј), and two different ␥ mixed DnaX complexes ( 1 ␥ 2 ␦␦Ј and 2 ␥ 1 ␦␦Ј) (15, 17, 19 -21). A novel assembly mechanism for the DnaX complex has been discovered recently; free DnaX is a tetramer in equilibrium with a free monomer (K D ϭ 170 nM), but the DnaX 4 stoichiometry is altered upon ␦␦Ј association, leading to the formation of a DnaX 3 ␦␦Ј complex (21). Both and ␥ are found co-assembled in purified holoenzyme and in pol III*, a subassembly of holoenzyme that lacks only ␤ (22, 23), but the assembly mechanism is not well understood. When the 10 subunits of holoenzyme are mixed simultaneously, they rapidly form a nine-subunit assembly containing but not ␥ (15). An alternative pathway through pol IIIЈ, an isolable subassembly comprised of 2 (pol III core) 2 (10), was also investigated, but the ␥ complex and pol IIIЈ did not associate upon mixing (17). If the entire complement of DnaX proteins is overexpressed from a single operon, ␥ mixed DnaX complexes are formed and can be purified by SP-Sepharose chromatography (21). Also, two in vitro protocols have been developed that produce a pol III* that contains both and ␥ in the same complex (17). In view of the important roles for the DnaX complex in replication and its unusual mechanism of assembly, we investigated the effects of various accessory proteins on the time course of the co-assembly of and ␥. We hoped to discover the factors required for the assembly of and ␥ into the same complex and to eventually dissect the steps in the assembly pathway. In addition, efficient in vitro assembly of a proper mixed complex will be useful in future studies on the roles of specific proteins and their interactions. We have determined that ␥ association proceeds slowly and the presence of the DnaX complex auxiliary proteins impedes this association. We looked at the assembly of and ␥ into pol III* and discovered that pol III core, in the absence of DnaX complex accessory proteins, stimulates co-assembly, suggesting that the holoen-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. 1 The abbreviations used are: holoenzyme, E. coli DNA polymerase III holoenzyme; pol III core, E. coli DNA polymerase core (␣⑀); ␥ mixed DnaX complex, 1 ␥ 2 ␦␦Ј or 2 ␥ 1 ␦␦Ј; ␥ complex, ␥ 3 ␦␦Ј; complex, 3 ␦␦Ј; pol III*, a complex containing all of the holoenzyme components except for ␤; pol IIIЈ, 2 (pol III core) 2 ; DTT, dithiothreitol.