The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC). The delta subunit of the gamma complex binds to the beta ring and opens it. The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, threedimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.DNA helicases are ubiquitous proteins that unwind doublestranded DNA (dsDNA) into single-stranded DNA (ssDNA), using energy from NTP hydrolysis (1). Interestingly, most of the helicases that have been studied to date self-assemble into either dimers or hexamers (2, 3). The significance of oligomerization in helicases is unclear, as the mechanism of DNA unwinding is not understood. Bacteriophage T7 gene 4 proteins provide the helicase and primase functions for T7 DNA replication and both proteins have been shown to form stable hexamers in the presence of Mg2+ and thymidine 5'-[I,3y-methylene]triphosphate (dTTP[3,), a nonhydrolyzable analog of dTTP (4). All data suggest that the hexamer is the active form of the protein involved in DNA unwinding, since hexamer formation is required for DNA binding, and substitution of a few of the hexameric subunits with inactive subunits inhibits both the DNA-dependent dTTPase and the helicase activities of the protein (5). We report here the structures of T7 gene 4A' and 4B proteins and their mode of DNA binding, using the techniques of electron microscopy, threedimensional reconstruction, and protein crosslinking. MATERIALS AND METHODSPreparation of Gene 4 Protein-DNA Complexes. T7 gene 4A' and 4B proteins were prepared as described (6). Preparations in the absence of DNA involved incubations of the protein (0.7 ,uM) for 10 min at 37°C in 25 mM triethanolamine buffer (pH 7.2) with 2 mM magnesium acetate and 1.3 mM dTTP[P3,y-CH2]. Preparations with ssDNA were similar, ex-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3869cept that the protein concentration was 1.5 ,uM and the DNA concentration was 0.6 nM.Synthesis of a-32P-Labeled-ssDNA. Radiolabeled linear ssDNA was prepared by elongating a 30-mer oligodeoxynucleotide with terminal deox...

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...

Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under presteady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s −1 at 20 °C) and then undergoes a slow step in the pathway that limits catalytic turnover (k cat = 0.1 s −1 ). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertioncontaining DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s −1 ). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 μM −1 s −1 ), indicating that for the Msh2-Msh6·mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatchinduced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway.Replicative DNA polymerases are responsible for accurately reproducing the genetic code of organisms; however, even the most accurate polymerases make errors that result in approximately one mismatched base pair per 10 7 nucleotides as well as insertion/deletion loops (1). These defects must be corrected prior to subsequent rounds of replication, to minimize accumulation of potentially deleterious mutations and genome instability. A multi-protein mismatch repair system is responsible for this task, which involves recognition and removal of the defects followed by replacement with correct DNA. This repair system was initially identified and studied extensively in bacteria, and homologous systems were discovered in a variety of other organisms, including humans (2). The critical role of DNA mismatch repair in maintaining genome and cellular integrity is highlighted by the links between defective repair protein function and predisposition to cancer (e.g., hereditary nonpolyposis colorectal cancer (3)).The repair process begins with DNA binding and mismatch/defect recognition by prokaryotic MutS or eukaryotic MutS homologue proteins. MutS/Msh 1 proteins then signal other proteins downstream in the pathway to initiate DNA excision. In bacteria, MutH endonuclease nicks the unmethylated new DNA strand at a GATC site, which is followed by UvrD/Helicase II and † This work was supported by a grant from the N.I.H. .