Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

Abstract

The DnaX complex (DnaX 3 ␦␦) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, ␤ 2 , onto DNA. The complex contains three DnaX subunits, which occur in two forms: and the shorter ␥, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant or ␥ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATPbinding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. ␤ 2 binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for ␤ 2 . DNA synthesis activity can be restored by increased concentrations of ␤ 2 . In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances ␤ 2 loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes. DNA polymerase III holoenzyme (pol III HE)2 exhibits features common to all other cellular replicases. It is a tripartite assembly composed of a replicative polymerase (pol III), a sliding clamp processivity factor (␤ 2 ), and an AAAϩ ATPase that assembles the ␤ 2 clamp around DNA (DnaX complex) (1, 2). The DnaX complex contains three DnaX subunits and one each of ␦, ␦Ј, , and . The DnaX subunit is either the full-length dnaX translation product or a shorter version, ␥, which is generated by translational frameshifting. ␥ lacks two C-terminal domains that bind the replicative helicase and pol III.Current models for clamp loading, largely derived from ␥-only DnaX complexes in the absence of pol III, propose that ATP binds to all three DnaX subunits, increasing the affinity of the clamp loader for ␤ 2 and primed DNA (1, 3). Once a ␤ 2 -primed DNA complex is formed, hydrolysis of the three ATPs places the DnaX complex into a conformation with decreased affinity for DNA, leading to its dissociation from DNA-bound ␤ 2 (3). pol III then associates with ␤ 2 in a downstream step, leading to formation of an initiation complex for processive replication.We have shown that some of the characteristics of initiation complex formation catalyzed by -containing DnaX complexes are different from ␥-only complexes. For example, complex, when bound to pol III, can form initiation complexes in the leading strand half of a dimeric replicase in the presence of the nonhydrolyzed an...

The ATP Sites of AAA+ Clamp Loaders Work Together as a Switch to Assemble Clamps on DNA

“…Because Rfc5 (E site) lacks a trans-acting Arg finger and contains mutations in the Walker B motif that are key for hydrolysis, this mutant was not investigated. Our study extends previous work measuring the effects of Walker A mutations on clamp loading and DNA synthesis in vitro and in vivo (11,12,14,15,43,44) by analyzing the effect of single site Walker A mutations on individual interactions and steps in the clamp loading reaction cycle.…”

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

“…DnaX complexes containing a mixture of inactive and wildtype DnaX subunits can function on long single-stranded templates in 5-min reactions (26). Examination of the kinetics of initiation complex formation reveals a 3500-fold lower rate for DnaX complexes containing a single inactive ATPase subunit (39).…”

“…The mutation occurs within the Walker A motif and abolishes ATP binding (26). In the presence of Pol III* containing mutant ␥, the total level of rolling circle DNA synthesis is decreased 7-fold (Fig.…”

“…(4). The purifications of the various forms of the DnaX complexes were performed by high-resolution FPLC chromotography on Mono S columns, providing baseline resolution between the forms with no cross-contamination, as revealed by rechromotography (26). D403E Pol III was assembled by incubating D403E Pol III ␣, ⑀, and at a molar ratio of 1:1:1 on ice for 15 min.…”

The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

“…A recent structural study with the T4 bacteriophage clamp loader suggested that hydrolysis of one ATP molecule causes a conformational change in the clamp loader⅐clamp complex that closes the clamp (30), and studies with the E. coli replisome suggest that hydrolysis of one ATP molecule is sufficient to form initiation complexes but hydrolysis of 3 molecules accelerates the process (31,32). Studies have shown that ATP hydrolysis is required for clamp release (24,26,33), but the question here is whether ATP hydrolysis is required for clamp closing or whether binding of the clamp loader⅐clamp complex to DNA is sufficient to promote clamp closing.…”