Rat polymerase beta binds double-stranded DNA using exclusively the 8-kDa domain. Stoichiometries, intrinsic affinities, and cooperativities.
Analyses of the interactions of rat polymerase beta (rat pol beta) with a double-stranded DNA have been performed using the quantitative fluorescence titration and fluorescence energy transfer techniques. The obtained results show that rat pol beta binds to dsDNA oligomers with the site-size of the enzyme-dsDNA complex n = 5 +/- 1 base pairs. The small site-size of the complex is a consequence of engagement of only the 8-kDa domain in intrinsic interactions with the dsDNA. This conclusion is directly supported by the fluorescence energy transfer between the single tryptophan residue on the 31-kDa domain and fluorescence acceptor located on the DNA. The dsDNA oligomer is bound at a distance of at least 55 A from the tryptophan, excluding the 31-kDa domain from any closed contact with the DNA. Moreover, in the complex with the dsDNA, the enzyme is bound in "open" conformational state. The intrinsic interactions are accompanied by a net release of about four to five ions. The net ion release is dominated by cations as a result of the exclusive engagement of the 8-kDa domain in interactions. Magnesium affects the net ion release through direct binding of Mg(2+) cations to the protein. Surprisingly, binding of rat pol beta to the dsDNA is characterized by strong positive cooperative interactions, a very different behavior from that previously observed for pol beta complexes with the ssDNA and gapped DNAs. Contrary to intrinsic affinities, cooperative interactions are accompanied by a net uptake of about three to five ions. Anions have a large contribution to the net ion uptake, indicating that cooperative interactions characterize protein-protein interactions. The significance of these results for the pol beta functioning in damaged-DNA recognition processes is discussed.
Department of Human Biological Chemistry and Genetics and the Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Maria J
MJ
Jezewska
Roberto
R
Galletto
Wlodzimierz
W
Bujalowski
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D008958:Models, Molecular; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D051381:Rats; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D046911:Macromolecular Substances; D014364:Tryptophan; D004247:DNA; D019951:DNA Polymerase beta