Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase beta in its different ssDNA...

The kinetics of human polymerase beta (pol beta) binding to the single-stranded DNA, in the (pol beta)(16) and (pol beta)(5) binding modes, that differ in the number of occluded nucleotide residues in the protein-DNA complexes, have been examined, using the fluorescence stopped-flow technique. This is the first determination of the mechanism of ssDNA recognition by human pol beta. Binding of the enzyme to the ssDNA containing fluorescein in the place of one of the nucleotides is characterized by a strong DNA fluorescence increase, providing the required signal to quantitatively examine the complex mechanism of ssDNA recognition. The experiments were performed with the ssDNA 20-mer, which engages the polymerase in the (pol beta)(16) binding mode and encompasses the total DNA-binding site of the enzyme, and with the 10-mer, which exclusively forms the (pol beta)(5) binding mode engaging only the 8-kDa domain of the enzyme. The obtained data and analyses indicate that the (pol beta)(16) formation occurs by a minimum four-step, sequential mechanism: (reaction: see text). Formation of the (pol beta)(5) binding mode proceeds with the same mechanism; however, both binding modes differ in the energetics of the partial reactions and the structure of the intermediates. Quantitative amplitude analysis, using the matrix projection operator approach, allowed us to determine molar fluorescence intensities of all intermediates relative to the fluorescence of the free DNA. The results indicate that (pol beta)(16) binding mode formation, which is initiated by the association of the 8-kDa domain with the DNA, is followed by subsequent intermediates stabilized by DNA binding to the 31-kDa domain. Comparison with the (pol beta)(5) binding mode formation indicates that transitions of the enzyme-DNA complex in both modes are induced at the interface of the 8-kDa domain and the DNA. The sequential nature of the mechanism indicates the lack of a conformational preequilibrium of the enzyme prior to ssDNA binding.