A Function for the ψ Subunit in Loading the Escherichia coli DNA Polymerase Sliding Clamp

Abstract

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, and , which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, ␥ 3 ␦␦, ␥ 3 ␦␦, and ␥ 3 ␦␦. The subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the subunit does not contribute directly to clamp loading in our assays lacking singlestranded DNA-binding protein. The subunit also increases the affinity of the clamp loader for the clamp in assays in which ATP␥S is substituted for ATP. Interestingly, the affinity of the ␥ 3 ␦␦ complex for ␤ is no greater in the presence than in the absence of ATP␥S. A role for in stabilizing or promoting ATPand ATP␥S-induced conformational changes may explain why large conformational differences were not seen in ␥ 3 ␦␦ structures with and without bound ATP␥S. The ␤ clamp partially compensates for the activity of when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.The efficiency of DNA replication is enhanced by processivity factors that enable a DNA polymerase to incorporate thousands of nucleotides in a single DNA-binding event. These processivity factors, a sliding clamp and a clamp loader, are conserved from bacteria to humans (recently reviewed in Refs. 1-4). Sliding clamps are ring-shaped protein complexes that encircle DNA, and the Escherichia coli clamp consists of two identical arc-shaped ␤ subunits (5). Clamps slide freely along duplex DNA so that a DNA polymerase bound to a clamp is tethered to the template being copied yet able to move along DNA at rates limited by the rate of nucleotide incorporation (6, 7). Clamp loaders catalyze the assembly of sliding clamps on DNA. The complete clamp loader at the E. coli replication fork contains two copies of the subunit and one copy each of the ␥, ␦, ␦Ј, , and subunits (8 -11). The and ␥ subunits are products of the same gene, dnaX, and the ␥ subunit is a truncated form produced during translation (12-14). Clamp loaders containing three copies of the DnaX protein in any combination are fully active in clamp loading (9), however, the additional C-terminal extension present on mediates protein-protein interactions that are required to coordinate other activities at the replication fork (reviewed in Refs. 4,15,and 16).The functional core of clamp loaders is composed of five subunits that are members of the AAAϩ family of ATPases (ATPases associated with diverse cellular activities; for recent AAAϩ reviews see Refs. 17 and 18).2 Three copies of the ␥ subunit, and one copy each of the ␦ and ␦Ј subu...

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of β Clamp Binding by the Escherichia coli γ Complex Clamp Loader

“…Synthetic oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA) and purified by 12% denaturing polyacrylamide gel electrophoresis. The sequences of the 60-nucleotide template and the complementary 30-nucleotide primer are as follows: 5Ј-TTC AGG TCA GAA GGG TTC TAT CTC TGT TGG CCA GAA TGT CCC TTT TAT TAC TGG TCG TGT-3Ј and 5Ј-ACA CGA CCA GTA ATA AAA GGG ACA TTC (C6dT) GG-3Ј, where C6dT is a T with a C6 amino linker that was covalently labeled with 7-diethylaminocoumarin-3-carboxylic acid succinimidyl ester (Invitrogen) as described (16,21). Primed templates were annealed by incubating the 30-nucleotide primer with the 60-nucleotide template at 85°C for 5 min and then allowing the solution to slowly cool to room temperature.…”

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…The Pseudomonas-type subunit was found to be considerably longer than its E. coli counterpart (282 versus 137 residues). Despite this, multiple-sequence alignments revealed three distinct conserved regions corresponding to helices ␣1 and ␣4 of the E. coli and a region at the N terminus known to interact with domain III of /␥ (7,96).…”

“…based on homology searches with the E. coli proteins (Table 1). Nevertheless, such searches could not locate a homolog of the holD gene, which encodes the subunit of the clamp loader complex in E. coli (7). A heterodimer formed between the and subunits binds to the clamp loader complex [(/␥) 3 ␦␦Ј] of DNA polymerase III through a region at the N terminus of (96,231).…”

“…The complex mediates the handoff of the primed template molecule from the DnaG primase via single-stranded DNA-binding protein (SSB) to the clamp loader complex ( 3 ␦␦Ј), thus representing a functional link between the primosome complex (DnaB-DnaG) and Pol III HE within the replisome (275). Originally thought to represent a simple "bridge" between the clamp loader complex and the subunit, the subunit has now been shown to play a significant role in loading of the ␤ clamp onto its DNA template in E. coli (7).…”

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

“…The subunit is also known, to facilitate transfer of an RNA primer from primase to the clamp loader (22). Furthermore, the subunit is required to stabilize the oligomeric structure of the clamp loader upon which the architecture of the replisome is built (23)(24)(25).…”