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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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A homology model of the B. subtilis clamp-loader complex, with δ, δ′ and τ subunits represented in green, blue and red, respectively. The Cτ domain of the τ subunit is represented in pink.
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gkt173-F2: A homology model of the B. subtilis clamp-loader complex, with δ, δ′ and τ subunits represented in green, blue and red, respectively. The Cτ domain of the τ subunit is represented in pink.

Mentions: Using the structural information described above, together with homology modelling techniques, a model for the B. subtilis clamp-loader complex was produced. Homology models of the individual B. subtilis clamp-loader subunits were generated using the I-TASSER automated protein structure prediction server. The structural templates used to model each individual protein were automatically selected by the server through sequence alignment against a database of proteins of known structure, with the higher homology sequences being chosen. The obtained δ and δ′ models were principally based on their E. coli equivalents, resulting in similar tertiary structures. The τ region corresponding to E. coli γ was modelled using this protein as the main template, resulting in a similar structure. The Cτ domain of τ was modelled separately, and the obtained structure resulted from the contribution of several different protein structures with considerable sequence homology, including a partial solution structure of the E. coli Cτ domain and the structures of DnaA (a protein involved in the initiation of DNA replication) from three different organisms. The γ equivalent region and Cτ were also combined using I-TASSER. The assembly of the individual modelled subunits was made by structural alignment with their equivalents within the E. coli clamp-loader crystal structure, assuming a similar topology and shape for both complexes. The obtained model is depicted in Figure 2. ESI-ion mobility-MS measurements were conducted on the clamp-loader complex. However, the high degree of structural collapse seen in the gas phase rendered these results of limited structural value (see Supplementary Figure S3). Such behaviour of protein complexes with relatively open structures has been described previously (36).Figure 2.


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)

A homology model of the B. subtilis clamp-loader complex, with δ, δ′ and τ subunits represented in green, blue and red, respectively. The Cτ domain of the τ subunit is represented in pink.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt173-F2: A homology model of the B. subtilis clamp-loader complex, with δ, δ′ and τ subunits represented in green, blue and red, respectively. The Cτ domain of the τ subunit is represented in pink.
Mentions: Using the structural information described above, together with homology modelling techniques, a model for the B. subtilis clamp-loader complex was produced. Homology models of the individual B. subtilis clamp-loader subunits were generated using the I-TASSER automated protein structure prediction server. The structural templates used to model each individual protein were automatically selected by the server through sequence alignment against a database of proteins of known structure, with the higher homology sequences being chosen. The obtained δ and δ′ models were principally based on their E. coli equivalents, resulting in similar tertiary structures. The τ region corresponding to E. coli γ was modelled using this protein as the main template, resulting in a similar structure. The Cτ domain of τ was modelled separately, and the obtained structure resulted from the contribution of several different protein structures with considerable sequence homology, including a partial solution structure of the E. coli Cτ domain and the structures of DnaA (a protein involved in the initiation of DNA replication) from three different organisms. The γ equivalent region and Cτ were also combined using I-TASSER. The assembly of the individual modelled subunits was made by structural alignment with their equivalents within the E. coli clamp-loader crystal structure, assuming a similar topology and shape for both complexes. The obtained model is depicted in Figure 2. ESI-ion mobility-MS measurements were conducted on the clamp-loader complex. However, the high degree of structural collapse seen in the gas phase rendered these results of limited structural value (see Supplementary Figure S3). Such behaviour of protein complexes with relatively open structures has been described previously (36).Figure 2.

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