Abstract
Interactions between the human DNA polymerase beta (pol beta) and a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence increase of the poly(dA) etheno-derivative (poly(depsilonA)) as a function of the binding density of pol beta-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol beta forms a complex with a ssDNA in which 16 nucleotides are occluded by the enzyme. In the second phase, transition to a complex where the polymerase occludes only 5 nucleotides occurs. Thus, human pol beta binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol beta)16 and the second as (pol beta)5 binding modes. The analyses of the enzyme binding to ssDNA have been performed using statistical thermodynamic models, which account for the existence of the two binding modes of the enzyme, cooperative interactions, and the overlap of potential binding sites. The importance of the discovery that human pol beta binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol beta, is discussed.
Highlights
Polymerase [1] is one of the four recognized DNAdirected polymerases of the eucaryotic nucleus: ␣, , ␦, and ⑀ (1–3)
We provide direct evidence that human pol  binds the ssDNA in two binding modes,[16] and[5], which differ in the number of occluded nucleotide residues in the complex
Statistical Thermodynamic Model for the Cooperative Binding of a Large Ligand in Two Different Binding Modes to a One-dimensional Infinite, Homogeneous Lattice—The simplest statistical thermodynamic model that describes the binding of a large ligand, which occludes a number of n nucleotides in the complex, to an infinite, homogeneous lattice is the McGhee-von Hippel model (29)
Summary
Reagents and Buffers—All chemicals were reagent grade. All solutions were made with distilled and deionized Ͼ18 M⍀ (Milli-Q Plus) water. Determination of the Thermodynamically Rigorous Isotherms of Human pol  Binding to Unmodified ssDNA Homopolymers Using the MCT Method—Determination of the interaction parameters for the human pol -unmodified nucleic acid complexes has been performed using the macromolecular competition titration (MCT) method, recently developed by us (23), using the 16-mer, d⑀A(p⑀A)[15], as a fluorescent reference nucleic acid. In this method, the fluorescent reference nucleic acid (d⑀A(p⑀A)15) at total concentration, NTR, is titrated with the protein in the presence of a competing nonfluorescent nucleic acid (e.g. poly(dA)) of total concentration, NTS. The theoretical formula for the degree of binding of human pol  on d⑀A(p⑀A)15, 16, has been derived in this work and is defined by Equation 11 (see “Results”)
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