Human DNA polymerase β recognizes single-stranded DNA using two different binding modes

Abstract

Interactions between the human DNA polymerase β (pol β) 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(dεA)) as a function of the binding density of pol β-nucleic acid complexes reveals the existence of two binding phases. In the first high affinity phase, pol β 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 β binds a ssDNA in two binding modes, which differ in the number of occluded nucleotide residues. We designate the first complex as (pol β)16 and the second as (pol β)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 β binds a ssDNA, using different binding modes, for the possible mechanistic model of the functioning of human pol β, is discussed.