Rat polymerase β gapped DNA interactions: Antagonistic effects of the 5′ terminal PO4- group and magnesium on the enzyme binding to the gapped DNAs with different ssDNA gaps

Maria J. Jezewska, Roberto Galletto, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO4- group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg+2 differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO4 - group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO4- group increases the stability of internal intermediates with the increasing length of the gap, Mg+2 decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg+2 effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme.

Original languageEnglish (US)
Pages (from-to)125-160
Number of pages36
JournalCell Biochemistry and Biophysics
Volume38
Issue number2
DOIs
StatePublished - May 1 2003

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Keywords

  • Binding modes
  • DNA polymerases
  • DNA repair
  • DNA replication
  • Fluorescence titrations
  • Protein-DNA interactions
  • Stopped-flow kinetics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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