The Mechanism of ATP-Dependent Primer-Template Recognition by a Clamp Loader Complex

Simonetta, K.R.; Kazmirski, Steven L.; Goedken, Eric R.; Cantor, Aaron J.; Kelch, Brian A.; McNally, Randall; Seyedin, Steven N.; Makino, Debora L.; O'Donnell, Mike; Kuriyan, John

doi:10.1016/j.cell.2009.03.044

Cited by 150 publications

(253 citation statements)

References 56 publications

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“…B, the longest product at each time point was plotted as a function of time, and the rate was determined by the slope of a line intersecting the first five points. 7,8,10, and 11, respectively; percentages of 67-mer were 0.2, 7.6, 26, and 19% for lanes 8,9,11, and 12, respectively; and percentages of 91-mer were 80, 92, 92, 66, 61, and 81% for lanes 7-12, respectively. Right, the three DNA templates (a-c) with 32 P-labeled primers (indicated by an asterisk) made as described under "Experimental Procedures."…”

Section: Discussionmentioning

confidence: 99%

“…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). binds (41), and binds to SSB through its C-terminal domain (18,36,37).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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%

Section: Discussionmentioning

confidence: 99%

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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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“…The ␥ complex loads ␤ on primed template junctions with a 3Ј recessed end. The duplex region of DNA sits inside the cap, stabilized by positively charged residues, and the single-stranded overhang bends out of the cap through the gap between ␦ and ␦Ј (19).…”

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confidence: 99%

The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

Hayner

Bloom

2013

Journal of Biological Chemistry

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Background:The ␥ complex loads the ring-shaped ␤ sliding clamp onto DNA. Results: The rate of ␤ clamp closing is faster than the rate of ␤ release on DNA, and the first turnover of ATP hydrolysis is faster than ␤ closing. Conclusion: Clamp closing occurs before clamp release but after a burst of ATP hydrolysis.Significance: These results demonstrate that clamp release around DNA is a two-step process.

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“…Conserved sensor-motif residues are in close proximity and are involved in nucleotide-state sensing and allosteric communication. This figure was generated based on the structure of the E. coli clamp loader (PDB entry 3glf; Simonetta et al, 2009). Distances are given in Å .…”

Section: Crystallization and Structure Determinationmentioning

confidence: 99%

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

Zeymer

Barends

Werbeck

et al. 2014

Acta Cryst D Biol Crystallogr

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ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotidebinding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg 2+ ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

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The Mechanism of ATP-Dependent Primer-Template Recognition by a Clamp Loader Complex

Cited by 150 publications

References 56 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

The β Sliding Clamp Closes around DNA prior to Release by the Escherichia coli Clamp Loader γ Complex

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

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