INTERACTIONS OF HUMAN REPLICATION PROTEIN-A WITH OLIGONUCLEOTIDES

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA b inding protein that is essential for eukaryotic DNA replication. In or der to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stran ded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated t hat RPA binds as a heterotrimer. Stoichiometric binding reactions moni tored by fluorescence quenching indicated that the binding site size o f human RPA is 30 nucleotides and that between 20-30 nucleotides of DN A directly interact with RPA. The binding of RPA to DNA of different l engths was systematically examined using deoxythymidine-containing oli gonucleotides. We found that the binding affinity of RPA for short oli gonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from similar to 7 x 10(7) M(-1) for oligo( dT)10 to similar to 1.5 x 10(10) M(-1) for oligo(dT)50. Human RPA bind s to oligonucleotides with low cooperativity; the cooperativity parame ter (omega) for RPA binding was estimated to be approximately 15.