ATP Binding to the Escherichia coli Clamp Loader Powers Opening of the Ring-shaped Clamp of DNA Polymerase III Holoenzyme

Abstract

The Escherichia coli ␥ complex serves as a clamp loader, catalyzing ATP-dependent assembly of ␤ protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the ␥ complex during clamp loading. We show that the energy from ATP binding to ␥ complex powers several initial events in the clamp loading pathway. The ␥ complex (␥ 2 ␦␦) binds two ATP molecules (one per ␥ subunit in the complex) with high affinity (K d ‫؍‬ 1-2.5 ؋ 10 Rapid and efficient duplication of genomic DNA depends on biomolecular machines known as DNA replicases. In diverse organisms these multicomponent biological machines exhibit varying degrees of complexity, including substantial differences in subunit composition. It is apparent, however, that certain mechanisms of action and the associated protein tools are conserved through evolution. One prominent example is the mechanism for processive DNA replication common among replicases from bacteriophage T4, Escherichia coli, yeast, and humans. Briefly, a clamp loader uses energy from ATP to assemble a circular protein clamp around DNA; the clamp, now topologically linked to DNA, tethers DNA polymerase to the template, and by sliding freely on duplex DNA it allows continuous replication of several thousand nucleotides without a dissociation event (reviewed in Refs. 1-3).The E. coli clamp loader, ␥ complex, is a composite of five different proteins, ␥, ␦, ␦Ј, , and , of which ␥ is the ATPbinding subunit essential for clamp loading (4 -7). The clamp, ␤, is a dimeric ring with a 35-Å inner diameter, large enough to encircle DNA (8). ␤ and ␥ complex form part of DNA polymerase III holoenzyme, the replicative DNA polymerase of E. coli. The holoenzyme assembly also includes a core polymerase (␣⑀), composed of ␣, the DNA polymerase (9), ⑀, the proofreading exonuclease (10, 11), and of unknown function (12, 13), as well as , a dimeric protein that holds together two polymerase cores and binds one ␥ complex (14 -16). The core polymerase is a nonprocessive, inefficient DNA polymerase that extends a primer by only 10 -20 nucleotides before dissociating from the template (17). When the ␤ clamp tethers the core polymerase to template DNA, however, the enzyme develops high processivity and extends DNA by several thousand nucleotides per binding event (18,19).In the current model for E. coli DNA replication, after the assembly of an initiation complex in which the polymerase III holoenzyme is tethered to a primed DNA template by circular protein clamps, the two core polymerases in the holoenzyme synthesize leading and lagging strand DNA synchronously (20 -23). Synthesis of the leading strand occurs in the direction of replication fork movement, but the lagging strand is synthesized in the opposite direction in discrete 1-2-kilobase pairlong Okazaki fragments. Therefore on the leading strand, after one clamp load...

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

“…The remaining two subunits, and , are involved in recruiting an RNA primed DNA site from the primase, and they bind singlestranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of ␥ complex (12)(13)(14). Biochemical studies of ␥ complex (15)(16)(17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex (18,19), reveal a highly detailed view of ␥ 3 ␦␦Ј clamp loader form and function. The five subunits of the ␥ 3 ␦␦Ј complex are arranged in a circular formation (see Fig.…”

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

“…A variety of steady-state and presteady-state techniques have been used to investigate the kinetic assembly of the holoenzyme in bacteriophage T4 (23)(24)(25) and in E. coli (26)(27)(28)(29)(30) Both systems are assembled stepwise to form their holoenzymes. In bacteriophage T4, gp44͞62 hydrolyzes two molecules of ATP to open gp45 and then two more molecules of ATP on interaction with DNA (14,23,24,31).…”

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

“…7, which is published as supporting information on the PNAS web site). The rms deviation in C ␣ positions for domain I of the ␥-subunits [excluding the zinc-binding loop (residues 64-79) and an N-terminal region (residues [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] that is structurally a part of domain II] is Ϸ1.5 Å at 2.0 ns, and the deviation in the corresponding segments of ␦ A and ␦Ј E is similar (Fig. 7).…”

“…ATP binding to the clamp loader triggers a conformational change that allows the complex to bind to and open the clamp and load it onto DNA (11,12). In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11,13) and separation of the clamp-loader complex from the DNA-loaded clamp.…”

“…In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11,13) and separation of the clamp-loader complex from the DNA-loaded clamp. How ATP binding is coupled to loading of the clamp on DNA is poorly understood, and elucidating the molecular steps of this process is of interest not only because of its central role in replication but also because of the widespread use of AAA ϩ ATPases in biology (8).…”