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

Interactions of rat DNA polymerase beta with a single-stranded (ss) DNA hav e been studied using the quantitative fluorescence titration technique. Exa mination of the fluorescence changes accompanying the binding, as a functio n of the thermodynamically rigorous binding density of rat pol beta-ssDNA c omplexes, reveals the existence of two binding phases. In the first high af finity phase, rat pol beta forms a complex with the ssDNA in which 16 nucle otides are occluded by the enzyme. In the second low affinity phase, a tran sition to a complex where the polymerase occludes only five nucleotides occ urs. Thus, the data show that rat pol beta binds the ssDNA in two binding m odes which differ in the number of occluded nucleotides. We designate the f irst complex as the (pol beta)(16) binding mode and the second as the (pol beta)(5) binding mode. The formation of the (pol beta)16 and (pol beta)(5) modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol beta)(16) or the (pol beta)(5) mode, and a 10mer which can form only the (pol beta)(5) mode. Binding of rat pol beta to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding mod es, cooperative interactions, and the overlap of potential binding sites. T he results indicate that the 8 kDa domain of the enzyme is involved in ssDN A 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 beta)(5) mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol beta )(5) mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions re leased, and a lower fluorescence change accompanying the formation of the ( pol beta)(16) than the (pol beta)(5) mode indicate that the 31 kDa catalyti c domain of the enzyme interacts with the ssDNA only in the (pol beta)(16) mode. The significance of these results for understanding the functioning o f rat pol beta in the DNA metabolism is discussed.