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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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Electrospray ionization-mass spectra of different combinations of the clamp-loader subunits from B. subtilis sprayed from 1 M ammonium acetate, pH7.5. The τ subunits are represented by red spheres, while the δ and δ′ subunits are represented by green and blue spheres, respectively. A, τ subunit in isolation; B, equimolar mixture of τ and δ subunits; C, equimolar mixture of τ and δ′ subunits; D, equimolar mixture of τ3, δ and δ′ subunits, resulting in the formation of the clamp-loader complex. E, The clamp-loader complex analysed under high collisional energy, resulting in a partial dissociation of δ and δ′ subunits (peaks corresponding to the low charged τ3-δ and τ3-δ′ after dissociation are magnified in the shaded region of the spectrum).
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gkt173-F1: Electrospray ionization-mass spectra of different combinations of the clamp-loader subunits from B. subtilis sprayed from 1 M ammonium acetate, pH7.5. The τ subunits are represented by red spheres, while the δ and δ′ subunits are represented by green and blue spheres, respectively. A, τ subunit in isolation; B, equimolar mixture of τ and δ subunits; C, equimolar mixture of τ and δ′ subunits; D, equimolar mixture of τ3, δ and δ′ subunits, resulting in the formation of the clamp-loader complex. E, The clamp-loader complex analysed under high collisional energy, resulting in a partial dissociation of δ and δ′ subunits (peaks corresponding to the low charged τ3-δ and τ3-δ′ after dissociation are magnified in the shaded region of the spectrum).

Mentions: To identify the oligomeric state adopted by the τ subunit from B. subtilis in isolation, a mass spectrum of the purified protein was recorded. Five charge-state distributions were observed corresponding to five different oligomers (Figure 1A), with the most abundant having a measured mass of 188 712 ± 12 Da. This value is in close agreement with the calculated mass of the τ trimer, 188 288 Da. The measured mass of the other four charge state distributions were found to correspond to the monomeric, dimeric, tetrameric and pentameric forms of τ. This result suggests a solution equilibrium between these five species.Figure 1.


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)

Electrospray ionization-mass spectra of different combinations of the clamp-loader subunits from B. subtilis sprayed from 1 M ammonium acetate, pH7.5. The τ subunits are represented by red spheres, while the δ and δ′ subunits are represented by green and blue spheres, respectively. A, τ subunit in isolation; B, equimolar mixture of τ and δ subunits; C, equimolar mixture of τ and δ′ subunits; D, equimolar mixture of τ3, δ and δ′ subunits, resulting in the formation of the clamp-loader complex. E, The clamp-loader complex analysed under high collisional energy, resulting in a partial dissociation of δ and δ′ subunits (peaks corresponding to the low charged τ3-δ and τ3-δ′ after dissociation are magnified in the shaded region of the spectrum).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt173-F1: Electrospray ionization-mass spectra of different combinations of the clamp-loader subunits from B. subtilis sprayed from 1 M ammonium acetate, pH7.5. The τ subunits are represented by red spheres, while the δ and δ′ subunits are represented by green and blue spheres, respectively. A, τ subunit in isolation; B, equimolar mixture of τ and δ subunits; C, equimolar mixture of τ and δ′ subunits; D, equimolar mixture of τ3, δ and δ′ subunits, resulting in the formation of the clamp-loader complex. E, The clamp-loader complex analysed under high collisional energy, resulting in a partial dissociation of δ and δ′ subunits (peaks corresponding to the low charged τ3-δ and τ3-δ′ after dissociation are magnified in the shaded region of the spectrum).
Mentions: To identify the oligomeric state adopted by the τ subunit from B. subtilis in isolation, a mass spectrum of the purified protein was recorded. Five charge-state distributions were observed corresponding to five different oligomers (Figure 1A), with the most abundant having a measured mass of 188 712 ± 12 Da. This value is in close agreement with the calculated mass of the τ trimer, 188 288 Da. The measured mass of the other four charge state distributions were found to correspond to the monomeric, dimeric, tetrameric and pentameric forms of τ. This result suggests a solution equilibrium between these five species.Figure 1.

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