Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA

Interactions between the isolated 8-kDa domain of the rat DNA polymerase be ta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residue s occluded in the native 8-kDa domain complex with the ssDNA (the site size ) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magn esium cation binding, as well as the dramatic changes in the monovalent sal t effect on the protein-ssDNA interactions in the presence of Mg2+, indicat es that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)(5) binding mode f ormed by an intact polymerase, indicating that the intact enzyme, but not t he isolated domain, has the ability to use only part of the domain DNA-bind ing site in its interactions with the nucleic acid. Salt effect on the intr insic interactions of the domain with the ssDNA indicates that a net releas e of m approximate to 5 ions accompanies the complex formation. Independenc e of the number of ions released upon the type of anion in solution strongl y suggests that the domain forms as many as seven ionic contacts with the s sDNA. Experiments with different ssDNA oligomers show that the affinity dec reases gradually with the decreasing number of nucleotide residues in the o ligomer. The data indicate a continuous, energetically homogeneous structur e of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the do main has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The sig nificance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.