Kinetic mechanisms of rat polymerase beta-ssDNA interactions. Quantitativefluorescence stopped-flow analysis of the formation of the (Pol beta)(16) and (Pol beta)(5) ssDNA binding mode

Kinetics of rat polymerase beta (pol beta) binding to the single-stranded D NA (ssDNA) in the (pol beta)(16) and (pol beta)(5) binding modes has been e xamined, using the fluorescence stopped-flow technique. Binding of the enzy me to the ssDNA containing fluorescein is characterized by a strong increas e of the DNA fluorescence, which provides an excellent signal to quantitati vely study the complex mechanism of the ssDNA recognition process. The expe riments were performed with a 20-mer ssDNA, which can engage the enzyme in the (pol beta)(16) binding mode, i.e. it encompasses the entire, total DNA- binding site of rat pol beta, and with a 10-mer which binds the enzyme excl usively in the (pol beta)(5) binding mode where only the 8 kDa domain of th e enzyme is engaged in interactions with the DNA. The data indicate that th e formation of the (pol beta)(16) binding mode occurs by a minimum three-st ep mechanism with the bimolecular binding step followed by two isomerizatio ns: Rat pol beta + ssDNA <----> (k1)(k-1) (P-16 - ssDNA)(1) <----> (k2)(k-2)(P-16 - ssDNA)(2) <----> (k3)(k-3)(P-16 - ssDNA)(3) A similar mechanism is observed in the formation of the (pol beta)(5) bindi ng mode, although at low salt concentrations there is an additional, slow s tep in the reaction. The data analysis was performed using the matrix proje ction operator technique, a powerful method to address stopped-flow kinetic s, particularly, amplitudes. The binding modes differ in the free energy ch anges of the partial reactions and ion effects on transitions between inter mediates that reflect different participation of the two structural domains . The formation of both binding modes is initiated by the fast association with the ssDNA through the 8 kDa domain, followed by transitions induced by interactions at the interface of the 8 kDa domain and the DNA. In the (pol beta)(16) binding mode, the subsequent intermediates are stabilized by the DNA binding to the DNA-binding subsite on the 31 kDa domain. The data indi cate that interactions of the ssDNA-binding subsite of the 8 kDa domain wit h the ssDNA, controlled by the ion binding, induce conformational transitio ns of the formed complexes in both binding modes. The sequential nature of the determined mechanisms indicates a lack of kinetically significant confo rmational equilibrium of rat pol beta, prior to ssDNA binding.