Mechanism of β Clamp Opening by the δ Subunit ofEscherichia coli DNA Polymerase III Holoenzyme

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This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (, ␦, and ␦) were identified. The dnaX gene produces only the full-length product, , and therefore differs from Escherichia coli dnaX that produces two proteins (␥ and ). Nonetheless, the A. aeolicus proteins form a ␦␦ complex. The dnaN gene encoding the ␤ clamp was identified, and the ␦␦ complex is active in loading ␤ onto DNA. A. aeolicus contains one dnaE homologue, encoding the ␣ subunit of DNA polymerase III. Like E. coli, A. aeolicus ␣ and interact, although the interaction is not as tight as the ␣؊ contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the ⑀ proofreading 3-5-exonuclease, interacts with ␣ but does not form a stable ␣⅐⑀ complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90°C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.Chromosomal replicases of all cellular organisms studied thus far are composed of three components, the DNA polymerase, a ring-shaped DNA sliding clamp, and a clamp loader that uses ATP to assemble the sliding clamp onto DNA (1-3). In bacteria, the sliding clamp is a homodimer called ␤ (4). The ring-shaped ␤ dimer completely encircles DNA and slides along the duplex (5). The ␤ clamp also binds the DNA polymerase III, thereby tethering it to DNA for high processivity (5).This report on the Aquifex aeolicus pol III 1 replicase is part of our continuing study of comparing and contrasting replicases from a variety of bacteria. Most knowledge of bacterial DNA polymerase III (pol III) structure and function has been obtained from studies of the Escherichia coli replicase, DNA polymerase III holoenzyme (reviewed in Ref. 6). Therefore, a brief overview of its structure and function is instructive for the comparisons to be made in this report. In E. coli, the catalytic subunit of DNA polymerase III is the ␣ subunit (129.9 kDa) encoded by dnaE; it lacks a proofreading exonuclease (7). The proofreading 3Ј-5Ј-exonuclease activity is contained in the ⑀ (27.5 kDa) subunit (dnaQ) that forms a 1:1 complex with ␣ (8, 9). The pol III ␣Ϫ⑀ complex is found tightly associated to a third subunit, called , to form the heterotrimeric E. coli DNA polymerase III core (10). The subunit (holE, 8.6 kDa) is not essential for growth and is generally not conserved in bacteria (11).The E. coli pol III ␣ subunit and pol III core subassembly act distributively on primed ssDNA and have only low activity; they are even further inhibited by the presence of SSB (7, 12). However, after the ␤ clamp has been assembled onto a primed s...

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confidence: 99%

Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile

Bruck¹,

Yuzhakov²,

Yurieva³

et al. 2002

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“…In the absence of ATP, the ␥ complex does not bind ␤ (18). Study of the ␦Ј subunit shows that it modulates the ability of ␦ to bind ␤ even in the absence of other ␥ complex subunits (17,19). Thus, ␦Ј is proposed to obscure the ␦ subunit within ␥ complex from binding to ␤ when ATP is not present (17,20).…”

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confidence: 99%

“…The ␦ subunit is referred to as the wrench of the clamp loader, since it can open the ␤ dimer at one interface on its own (17)(18)(19)(20). The energy for ring opening is not derived from ATP (neither ␦ nor ␤ bind ATP) but from the energy of protein-protein interaction between ␦ and ␤ (17).…”

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confidence: 99%

Mechanism of the δ Wrench in Opening the β Sliding Clamp

O'Donnell

2003

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The ␤ sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, ␥ complex (␥ 3 ␦␦), assembles ␤ around DNA in an ATP-fueled reaction. The ␦ subunit of the clamp loader opens the ␤ ring and is referred to as the wrench; ATP modulates contact between ␤ and ␦ among other functions. Crystal structures of ␦⅐␤ and the ␥ 3 ␦␦ minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The ␦ wrench contacts ␤ at two sites. One site is at the ␤ dimer interface, where ␦ appears to distort the interface by via a steric clash between a helix on ␦ and a loop near the ␤ interface. The energy for this steric clash is thought to derive from the other site of interaction, in which ␦ binds to a hydrophobic pocket in ␤. The current study demonstrates that rather than a simple steric clash with ␤, ␦ specifically contacts ␤ at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of ␦ at the hydrophobic site on ␤ contributes to destabilization of the ␤ dimer interface rather than acting solely as a grip of ␦ on ␤. Within the ␥ complex, ␦ is proposed to prevent ␦ from binding to ␤ in the absence of ATP. This report demonstrates that one or more ␥ subunits also contribute to this role. The results also indicate that ␦ acts as a backboard upon which the ␥ subunits push to attain the ATP induced change needed for the ␦ wrench to bind and open the ␤ ring.

“…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.…”

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confidence: 99%

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

Yao

Coryell

Zhang

et al. 2003

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Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, ␥ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA؉ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli ␥ complex in which only one subunit makes strong contact with the ␤ clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric ␤ ring.Replicases of cellular chromosomes utilize a circular sliding clamp protein that encircles DNA and tethers the polymerase to the template for high processivity in DNA synthesis (1). An example of this protein class is the Escherichia coli ␤ subunit, which confers high processivity onto the chromosomal replicase, DNA polymerase III holoenzyme (2, 3). The eukaryotic equivalent is the PCNA 1 ring, which has essentially the same shape and chain fold as ␤ despite lack of sequence similarity between the two (4, 5). These ring-shaped proteins require an ATP-fueled multiprotein clamp loader for assembly onto primed DNA.The eukaryotic clamp loader is the heteropentameric replication factor C (RFC). The five subunits of RFC are each different proteins, but they are homologous to one another (6, 7) and are members of the AAAϩ family of ATPases (8). The recent crystal structure of E. coli ␥ complex, the prokaryotic counterpart of RFC, has facilitated detailed hypothesis regarding RFC structure and mechanism (9, 10). The ␥ complex (␥ 3 ␦␦Ј ) consists of a minimal core of five proteins (␥ 3 ␦␦Ј), which contain the clamp loading activity (11). 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 (15-17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex ...