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Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

Afonso JP, Chintakayala K, Suwannachart C, Sedelnikova S, Giles K, Hoyes JB, Soultanas P, Rafferty JB, Oldham NJ - Nucleic Acids Res. (2013)

Bottom Line: The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions.Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli.Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

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Related in: MedlinePlus

The crystal structure of B. subtilis δ (yqeN). (A) Cartoon representation of the overall structure of δ. Domain I (residues 1–143), Domain II (residues 144–214) and Domain III (residues 215–339) are coloured magenta, green and blue, respectively. (B) Cartoon representation of δ, with α-helices, β-strands and loops shown as cyan cylinders, red arrows and magenta lines, respectively. (C) Topology diagram of δ with individual elements and residues labelled for α-helices (cyan cylinders) and β-sheets (red arrows) with connecting loops (magenta lines). Produced using TOPDRAW from the CCP4 suite.
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gkt173-F3: The crystal structure of B. subtilis δ (yqeN). (A) Cartoon representation of the overall structure of δ. Domain I (residues 1–143), Domain II (residues 144–214) and Domain III (residues 215–339) are coloured magenta, green and blue, respectively. (B) Cartoon representation of δ, with α-helices, β-strands and loops shown as cyan cylinders, red arrows and magenta lines, respectively. (C) Topology diagram of δ with individual elements and residues labelled for α-helices (cyan cylinders) and β-sheets (red arrows) with connecting loops (magenta lines). Produced using TOPDRAW from the CCP4 suite.

Mentions: To assess the accuracy of the clamp-loader model described above, the crystal structure of δ (coded by the B. subtilis yqeN gene) was determined at 2.1 Å resolution. It revealed a protein composed of three domains: domain I (residues 1–143), domain II (residues 144–214) and domain III (residues 215–339). The overall structure and the topology diagram of δ are shown in Figure 3. The N-terminal domain I consists of a central five-stranded parallel β sheet (β1–β5) surrounded by seven α-helices (α1–α7). This domain has a RecA-like fold that resembles the core of the nucleotide binding domain of RecA (37). Domain II is composed of just four α-helices (α8–α11). Finally, the C-terminal domain III is also predominantly helical and consists of seven α-helices (α12–α18). Examination of the crystal packing of the protein confirms the monomeric state observed from gel filtration during protein purification, and the overall surface area of δ calculated by the PISA server (38) was ∼17 000 Å2.Figure 3.


Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

Afonso JP, Chintakayala K, Suwannachart C, Sedelnikova S, Giles K, Hoyes JB, Soultanas P, Rafferty JB, Oldham NJ - Nucleic Acids Res. (2013)

The crystal structure of B. subtilis δ (yqeN). (A) Cartoon representation of the overall structure of δ. Domain I (residues 1–143), Domain II (residues 144–214) and Domain III (residues 215–339) are coloured magenta, green and blue, respectively. (B) Cartoon representation of δ, with α-helices, β-strands and loops shown as cyan cylinders, red arrows and magenta lines, respectively. (C) Topology diagram of δ with individual elements and residues labelled for α-helices (cyan cylinders) and β-sheets (red arrows) with connecting loops (magenta lines). Produced using TOPDRAW from the CCP4 suite.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643586&req=5

gkt173-F3: The crystal structure of B. subtilis δ (yqeN). (A) Cartoon representation of the overall structure of δ. Domain I (residues 1–143), Domain II (residues 144–214) and Domain III (residues 215–339) are coloured magenta, green and blue, respectively. (B) Cartoon representation of δ, with α-helices, β-strands and loops shown as cyan cylinders, red arrows and magenta lines, respectively. (C) Topology diagram of δ with individual elements and residues labelled for α-helices (cyan cylinders) and β-sheets (red arrows) with connecting loops (magenta lines). Produced using TOPDRAW from the CCP4 suite.
Mentions: To assess the accuracy of the clamp-loader model described above, the crystal structure of δ (coded by the B. subtilis yqeN gene) was determined at 2.1 Å resolution. It revealed a protein composed of three domains: domain I (residues 1–143), domain II (residues 144–214) and domain III (residues 215–339). The overall structure and the topology diagram of δ are shown in Figure 3. The N-terminal domain I consists of a central five-stranded parallel β sheet (β1–β5) surrounded by seven α-helices (α1–α7). This domain has a RecA-like fold that resembles the core of the nucleotide binding domain of RecA (37). Domain II is composed of just four α-helices (α8–α11). Finally, the C-terminal domain III is also predominantly helical and consists of seven α-helices (α12–α18). Examination of the crystal packing of the protein confirms the monomeric state observed from gel filtration during protein purification, and the overall surface area of δ calculated by the PISA server (38) was ∼17 000 Å2.Figure 3.

Bottom Line: The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions.Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli.Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

Show MeSH
Related in: MedlinePlus