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.
Jezewska
Maria J
MJ
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.
Galletto
Roberto
R
Bujalowski
Wlodzimierz
W
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