Interactions of the 8-kDa domain of rat DNA polymerase β with DNA

M. J. Jezewska, S. Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Interactions between the isolated 8-kDa domain of the rat DNA polymerase β and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 ± 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 ± 0.6 nucteotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein - ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain - ssDNA complex is significantly larger than the 5 ± 2 site size determined for the (pol β)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m ≈ 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol β is discussed.

Original languageEnglish (US)
Pages (from-to)3295-3307
Number of pages13
JournalBiochemistry
Volume40
Issue number11
DOIs
StatePublished - Mar 20 2001

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DNA-Directed DNA Polymerase
Rats
Binding Sites
DNA
Nucleotides
Oligomers
Cations
Salts
Ions
Titration
Magnesium
Nucleic Acids
Anions
Conformations
Proteins
Fluorescence
Enzymes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interactions of the 8-kDa domain of rat DNA polymerase β with DNA. / Jezewska, M. J.; Rajendran, S.; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 40, No. 11, 20.03.2001, p. 3295-3307.

Research output: Contribution to journalArticle

Jezewska, M. J. ; Rajendran, S. ; Bujalowski, Wlodzimierz. / Interactions of the 8-kDa domain of rat DNA polymerase β with DNA. In: Biochemistry. 2001 ; Vol. 40, No. 11. pp. 3295-3307.
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abstract = "Interactions between the isolated 8-kDa domain of the rat DNA polymerase β and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 ± 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 ± 0.6 nucteotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein - ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain - ssDNA complex is significantly larger than the 5 ± 2 site size determined for the (pol β)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m ≈ 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol β is discussed.",
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