The N-terminal domain of the escherichia coli pria helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain-DNA interactions and allosteric effect of the nucleotide cofactors

Michal R. Szymanski, Pawel Bujalowski, Maria J. Jezewska, Aleksandra M. Gmyrek, Wlodzimierz Bujalowski

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Abstract

Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein-double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer-single-stranded DNA (ssDNA) complex is ∼13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain-N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

Original languageEnglish (US)
Pages (from-to)9167-9183
Number of pages17
JournalBiochemistry
Volume50
Issue number43
DOIs
StatePublished - Nov 1 2011

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Escherichia coli
Nucleotides
Binding Sites
Dimers
Adenosine Diphosphate
Single-Stranded DNA
Conformations
Monomers
DNA
Enzymes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "The N-terminal domain of the escherichia coli pria helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain-DNA interactions and allosteric effect of the nucleotide cofactors",
abstract = "Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein-double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer-single-stranded DNA (ssDNA) complex is ∼13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain-N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.",
author = "Szymanski, {Michal R.} and Pawel Bujalowski and Jezewska, {Maria J.} and Gmyrek, {Aleksandra M.} and Wlodzimierz Bujalowski",
year = "2011",
month = "11",
day = "1",
doi = "10.1021/bi201100k",
language = "English (US)",
volume = "50",
pages = "9167--9183",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "43",

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TY - JOUR

T1 - The N-terminal domain of the escherichia coli pria helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain-DNA interactions and allosteric effect of the nucleotide cofactors

AU - Szymanski, Michal R.

AU - Bujalowski, Pawel

AU - Jezewska, Maria J.

AU - Gmyrek, Aleksandra M.

AU - Bujalowski, Wlodzimierz

PY - 2011/11/1

Y1 - 2011/11/1

N2 - Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein-double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer-single-stranded DNA (ssDNA) complex is ∼13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain-N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

AB - Functional interactions of the Escherichia coli PriA helicase 181N-terminal domain with the DNA and nucleotide cofactors have been quantitatively examined. The isolated 181N-terminal domain forms a stable dimer in solution, most probably reflecting the involvement of the domain in specific cooperative interactions of the intact PriA protein-double-stranded DNA (dsDNA) complex. Only one monomer of the domain dimer binds the DNA; i.e., the dimer has one effective DNA-binding site. Although the total site size of the dimer-single-stranded DNA (ssDNA) complex is ∼13 nucleotides, the DNA-binding subsite engages in direct interactions with approximately five nucleotides. A small number of interacting nucleotides indicates that the DNA-binding subsites of the PriA helicase, i.e., the strong subsite on the helicase domain and the weak subsite on the N-terminal domain, are spatially separated in the intact enzyme. Contrary to current views, the subsite has an only slight preference for the 3-end OH group of the ssDNA and lacks any significant base specificity, although it has a significant dsDNA affinity. Unlike the intact helicase, the DNA-binding subsite of the isolated domain is in an open conformation, indicating the presence of the direct helicase domain-N-terminal domain interactions. The discovery that the 181N-terminal domain possesses a nucleotide-binding site places the allosteric, weak nucleotide-binding site of the intact PriA on the N-terminal domain. The specific effect of ADP on the domain DNA-binding subsite indicates that in the intact helicase, the bound ADP not only opens the DNA-binding subsite but also increases its intrinsic DNA affinity.

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U2 - 10.1021/bi201100k

DO - 10.1021/bi201100k

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VL - 50

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JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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