Interactions of Human Replication Protein A with Oligonucleotides

Abstract

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA•DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20–30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from ∼7 × 107 M−1 for oligo(dT)10 to ∼1.5 × 1010 M−1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (ω) for RPA binding was estimated to be approximately 15. © 1994, American Chemical Society. All rights reserved.