The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes

Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known highaffinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1^DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1^DBD forms a highaffinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1^DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1^DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1^DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1^DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation. (Figure Presented).