Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

Michal R. Szymanski, Maria J. Jezewska, Wlodzimierz Bujalowski

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Abstract

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.

Original languageEnglish (US)
Pages (from-to)8-25
Number of pages18
JournalJournal of Molecular Biology
Volume398
Issue number1
DOIs
StatePublished - Apr 2010

Fingerprint

Single-Stranded DNA
Escherichia coli
Proteins
Nucleic Acids
Binding Sites
Ion Exchange
Ultracentrifugation
DNA
Entropy
Statistical Models
Thermodynamics
Anions
Nucleotides
Fluorescence
Ions

Keywords

  • DNA-binding
  • E. coli PriB protein
  • Fluorescence titrations
  • Primosome

ASJC Scopus subject areas

  • Molecular Biology

Cite this

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title = "Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities",
abstract = "Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.",
keywords = "DNA-binding, E. coli PriB protein, Fluorescence titrations, Primosome",
author = "Szymanski, {Michal R.} and Jezewska, {Maria J.} and Wlodzimierz Bujalowski",
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T1 - Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

AU - Szymanski, Michal R.

AU - Jezewska, Maria J.

AU - Bujalowski, Wlodzimierz

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N2 - Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.

AB - Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.

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