Faithful genome replication is carried out at the replication fork where the replisome synthesizes copies of each strand of native DNA. Due to the size of the genome in higher eukaryotes and the need for rapid cell division, replicative DNA polymerases ? and ? have evolved for tightly controlled, high-fidelity synthesis of the complementary strand. However, exposure to environmental mutagens, such as oxidizing chemicals or UV light may generate damage lesions that cause these polymerases and the full replication fork to stall. When a replicative polymerase encounters this damage on single-stranded DNA (ssDNA), the inability to continue synthesis causes the leading and lagging strand of the replication fork to become unsynchronized. This halts replication and leads to uncoupling of the polymerases and helicases, a phenomenon associated with diseases of genomic instability such as neurodegeneration and cancer. As a consequence of this fork collapse, long stretches of exposed ssDNA are coated with the ssDNA binding protein Replication Protein A (RPA), which serves as a signal to checkpoint proteins that initiate cell cycle arrest and recruits DNA damage response factors. One mechanism to recover from this stalled state involves the recruitment of a lower-fidelity polymerase that can bypass the lesion. Once the lesion is passed, the substrate is handed-off to Pol ? or Pol ? to restart fork progression, a process known as translesion synthesis (TLS). Recent studies have identified a novel TLS polymerase in humans named PrimPol for its intrinsic primase and polymerase activities. It was also discovered that PrimPol interacts with RPA, suggesting a possible mechanism for its recruitment to the collapsed replication fork. However, very little is known about the molecular details of this interaction or the functional role of RPA in PrimPol TLS catalytic activity. I hypothesize that the PrimPol-RPA interaction is necessary for PrimPol recruitment to ssDNA template and RPA displacement prior to TLS primer extension, and that RPA plays a differential regulatory role on the leading and lagging strand of the replication fork. This proposal will use a variety of structural, biophysical, and biochemical experiments to investigate the influence RPA on the ability of PrimPol to generate and extend primers. Understanding the molecular details of the PrimPol-RPA interface will allow for the development of accurate models of PrimPol-mediated TLS. More generally, it will provide insight into the observed ability of RPA to functionally distinguish proteins involved in DNA repair from those in processive replication. This information will aid in understanding the molecular basis of replication restart by TLS enzymes, as well as laying the mechanistic foundations of how the interactions of other DNA repair enzymes with RPA are essential to prevent genomic instability and carcinogenesis.