Dynamics of gapped DNA recognition by human polymerase β

Maria J. Jezewska, Roberto Galletto, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Kinetics of human polymerase β binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ∼600-1200 s-1. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5′-terminal PO4 - group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.

Original languageEnglish (US)
Pages (from-to)20316-20327
Number of pages12
JournalJournal of Biological Chemistry
Volume277
Issue number23
DOIs
StatePublished - Jun 7 2002

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DNA
Substrates
Enzymes
Association reactions
Single-Stranded DNA
Isomerization
Free energy
Rate constants
Nucleotides
Fluorescence
Phosphates
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dynamics of gapped DNA recognition by human polymerase β. / Jezewska, Maria J.; Galletto, Roberto; Bujalowski, Wlodzimierz.

In: Journal of Biological Chemistry, Vol. 277, No. 23, 07.06.2002, p. 20316-20327.

Research output: Contribution to journalArticle

Jezewska, Maria J. ; Galletto, Roberto ; Bujalowski, Wlodzimierz. / Dynamics of gapped DNA recognition by human polymerase β. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 23. pp. 20316-20327.
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