PCNA encircling primer/template junctions is eliminated by exchange of RPA for Rad51: implications for the interplay between human DNA damage tolerance pathways.

The DNA genome is constantly exposed to agents, such as ultraviolet radiation (UVR), that can alter or eliminate its coding properties through covalent modifications of the template bases. Many of these damaging modifications (i.e. lesions) persist into the S-phase of the cell cycle, where they may stall the canonical DNA replication machinery. In humans, these stalling events are circumvented by one of at least three interconnected DNA damage tolerance (DDT) pathways: translesion DNA synthesis (TLS), Template Switching (TS), and Homology-dependent Recombination (HDR). Currently, the functional interplay between these pathways is unclear, leaving wide gaps in our fundamental understanding of human DDT. To gain insights, we focus on the activation mechanisms of the DDT pathways. PCNA sliding clamps encircling primer/template junctions of stalled replication sites are central to the activation of both TLS and TS, whereas exchange of RPA for Rad51 filaments on the single-strand DNA (ssDNA) sequences of stalled replication sites is central to HDR activation. Utilizing direct, ensemble FRET approaches developed by our lab, we independently monitor and directly compare PCNA occupancy and RPA/Rad51 exchange at primer/template junctions under a variety of conditions that mimic in vivo scenarios. Collectively, the results reveal that assembly of stable Rad51 filaments at primer/template junctions via RPA/Rad51 exchange causes complete and irreversible unloading of the resident PCNA, both in the presence and absence of an abundant PCNA-binding protein complex. Further investigations decipher the mechanism of RPA/Rad51 exchange-dependent unloading of PCNA. Collectively, these studies provide critical insights into the interplay between human DDT pathways and direction for future studies.