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

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.