Abstract
The heterotrimeric Replication Protein A (RPA) complex preserves genome integrity by protecting the single-stranded DNA that becomes exposed during repair, replication, and recombination. Its two biggest subunits, Rfa1p and Rfa2p (as named in S. cerevisiae) contact DNA and interact with other partners, while the smallest Rfa3p subunit is considered to fulfill a structural role. Perhaps because of this, mostly Rfa1p and eventually Rfa2p are used for microscopy studies upon tagging them with fluorophores. In this work, we explore the behavior of GFP-tagged Rfa3p basally and in response to DNA damage conditions and compare it with tagged Rfa1p. We find that fluorescent Rfa3p yields signals that are (or are detected) significantly more frequent(ly). By making a careful comparison with our own and with previously published data, we propose that Rfa3p, by virtue of its scaffolding role, may reach single-stranded DNA sites first thus serving to nucleate the full RPA complex.