Transition between different binding modes in rat DNA polymerase β-ssDNA complexes

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

    Research output: Contribution to journalArticle

    36 Scopus citations

    Abstract

    Interactions of rat DNA polymerase β with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol β-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol β forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol β binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol β)16 binding mode and the second as the (pol β)5 binding mode. The formation of the (pol β)16 and (pol β)5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol β)16 or the (pol β)5 mode, and a 10mer which can form only the (pol β)5 mode. Binding of rat pol β to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol β)5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol β)5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol β)16 than the (pol β)5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol β)16 mode. The significance of these results for understanding the functioning of rat pol β in the DNA metabolism is discussed.

    Original languageEnglish (US)
    Pages (from-to)1113-1131
    Number of pages19
    JournalJournal of Molecular Biology
    Volume284
    Issue number4
    DOIs
    StatePublished - Dec 11 1998

    Keywords

    • DNA polymerase
    • DNA replication and repair
    • Protein-ssDNA interactions
    • Quantitative fluorescence titrations

    ASJC Scopus subject areas

    • Structural Biology
    • Molecular Biology

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