A Bipartite Polymerase–Processivity Factor Interaction: Only the Internal β Binding Site of the α Subunit is Required for Processive Replication by the DNA Polymerase III Holoenzyme

Dohrmann, Paul R.; McHenry, Charles S.

doi:10.1016/j.jmb.2005.04.065

Cited by 94 publications

(140 citation statements)

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“…The molecular interactions behind this protein network are well understood. Pol III binds in an interaction between the C terminus of Pol III and domain V of (12,39,40). One protomer binds a trimeric assembly of DnaX proteins through their domain III (5,11).…”

Section: Discussionmentioning

confidence: 99%

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Yuan

McHenry

2009

Journal of Biological Chemistry

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In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the singlestranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of ␥-complex to support the reaction in the absence of . However, if ␥-complex is present to load ␤ 2 , a truncated protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the ␣ subunit of DNA polymerase (Pol) III and . This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where is only required to serve as a scaffold to hold Pol III and in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476 -23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III----SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k cat /K m ) for the strand displacement reaction is ϳ300-fold less favorable than reactions on singlestranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (ϳ300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.All cellular replicases are tripartite assemblies, consisting of a replicative polymerase (Pol 2 III in bacteria, Pol ␦ or ⑀ in eukaryotes), a sliding clamp processivity factor (␤ 2 in bacteria, proliferating cell nuclear antigen in eukaryotes), and a clamp loader composed of a five-protein core of AAA ϩ -like proteins that assembles the sliding clamp around DNA (DnaX complex in bacteria, RFC in eukaryotes) (1-3). The Escherichia coli DnaX complex comprises three copies of DnaX and one each of ␦, ␦Ј, and (4 -6). E. coli and many other bacteria contain two forms of DnaX, the full-length translation product and a shorter protein, ␥, that results from translational frameshifting (7-9). Both and ␥ contain the three domains that are required for ATP-dependent ␤ 2 loading onto DNA (6). The third domain of and ␥ is responsible for binding other DnaX subunits as well as ␦, ␦Ј, and (5, 10, 11). contains two additional domains that interact with the DnaB replicative helicase (domain IV) and Pol III (domain V) (12, 13).The primary determinant of processivity of the E. coli replicase is the interaction of Pol III with ␤ 2 (14, 15). However, other interactions stabilize the interaction of Pol III with the replication fork. Two protomers bind the DnaB helicase, further stabilizing the replicase at the fork...

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Section: Discussionmentioning

confidence: 99%

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Yuan

McHenry

2009

Journal of Biological Chemistry

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“…Studies have identified two sequences within the Pol III ␣ subunit that bind to ␤; one is located at the extreme C terminus of ␣ (15), and the other is an internal site 220 residues upstream of the ␣ C terminus (16). The two sequences differ to some extent, but both fall within the conserved consensus sequence defined for ␤-binding peptides (9).…”

Section: Identification Of a Small-molecule Compound That Binds The Pmentioning

confidence: 99%

Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

Georgescu

Yurieva

Kim

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

154

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DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli ␤-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with ␤ and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to ␤. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with ␤, and that the ␤-clamp may represent an antibiotic target.antibiotic target ͉ rational drug design ͉ fluorescence anisotropy ͉ crystallography T he replication machinery of all cells utilizes a ring-shaped, sliding-clamp protein that encircles DNA and slides along the duplex, thus acting as a mobile tether to hold the chromosomal replicase to DNA for high processivity (1-3). Sliding clamps from the three domains of life are remarkably similar in architecture (4-6). They consist of six domains of similar chain fold. The bacterial ␤-clamp is a homodimer, and each protomer consists of three domains, whereas eukaryotic and archaeal proliferating cell nuclear antigen (PCNA) clamps are homotrimers formed from protomers containing two domains each.Initially, ␤ and PCNA were identified as processivity factors for chromosomal replicases, but sliding clamps are now known to function with diverse DNA polymerases, repair factors, and cell cycle-control proteins (reviewed in ref.

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“…E. coli ␣-polymerase subunit contains two regions that bind ␤ (62). The extreme C-terminal sequence binds the hydrophobic site on ␤, and the second region is about 200 residues internal to the C terminus (53,62). Mutations in either region alter the affinity of ␣ for ␤.…”

Section: Protein Trafficking On Clampsmentioning

confidence: 99%

Replisome Architecture and Dynamics in Escherichia coli

O'Donnell

2006

Journal of Biological Chemistry

126

114

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The field of DNA replication was launched upon discovery of the first DNA polymerase by Kornberg and Lehman (1,2). DNA polymerase I displayed the novel and highly exciting property of template-directed enzymatic action, and like a split personality, DNA polymerase I could also degrade DNA, from either direction. Escherichia coli is now known to contain five different DNA polymerases. The chromosomal replicase is DNA polymerase III, while the damage-inducible DNA polymerases II, IV, and V play roles in DNA repair. DNA polymerase I is involved in both replication and repair and remains the most advanced model for the structure and function of DNA polymerase action.DNA polymerase III (pol III) 2 functions in the context of a multiprotein apparatus called DNA pol III holoenzyme (reviewed in (3-5)). pol III holoenzyme contains 10 different proteins, which assort into three major functional units: 1) pol III core, 2) the ␤ sliding clamp, and 3) the ␥/ complex clamp loader. The holoenzyme contains two copies of the pol III core, which are connected by the attachment to one clamp loader. The holoenzyme functions within the context of a dynamic replisome containing pol III holoenzyme, a hexameric DnaB helicase, DnaG primase, and SSB. During replisome function, contacts between these proteins are in a constant state of change. This review briefly summarizes the architecture and dynamic behavior of the E. coli replisome. The Clamp and Clamp LoaderThe ␤ sliding clamp is a homodimer in the shape of a ring, which encircles DNA (Fig. 1A). The ␤ clamp slides on DNA and binds the pol III core, thereby acting as a mobile tether and converting the normally distributive pol III core into a highly processive and rapid polymerase capable of incorporating 500 -1,000 nucleotides per second (Fig. 1C). The crystal structure of the ␤ dimer reveals a 6-fold pseudo symmetry that arises from a domain that is repeated three times in the monomer giving the dimer a 6-fold appearance (6). The eukaryotic PCNA clamp and phage T4 gp45 clamp are also six-domain rings, but the monomeric unit contains only two domains and trimerizes to form a six-domain ring (7).Clamps do not self-assemble onto DNA but require a multiprotein clamp loader, which harnesses the energy of ATP hydrolysis to open and close the clamp around DNA (Fig. 1B). The E. coli ␥ complex clamp loader contains five subunits that are essential for clamp loading activity, three ␥ protomers and one copy each of ␦ and ␦Ј. The small and subunits are not required for clamp loading, but they stabilize the clamp loader and stimulate its activity at elevated ionic strength.The ␥, ␦, and ␦Ј clamp loading subunits are members of the AAAϩ family (ATPases associated with a variety of cellular functions) (8). The subunits consist of three domains, and a AAAϩ region of homology is localized to the first two domains (9). Many AAAϩ proteins are homohexamers arranged in a symmetric circle (10, 11). The ␥ 3 ␦␦Ј pentamer forms an asymmetric circle, and there is a gap in place of the "missing sixth subunit"...

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A Bipartite Polymerase–Processivity Factor Interaction: Only the Internal β Binding Site of the α Subunit is Required for Processive Replication by the DNA Polymerase III Holoenzyme

Cited by 94 publications

References 49 publications

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template

Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

Replisome Architecture and Dynamics in Escherichia coli

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