Some DNA viruses utilize cellular DNA damage response (DDR) pathways to aid in viral DNA replication (e.g. HSV and HPV), while others may attenuate the DDR response (e.g. adenovirus), or even be subject to DNA replication arrest by DDR (e.g. EBV and polyomavirus (PyV)). PyV DNA replication has been an important simplified model of host cell DNA replication that utilizes a single viral protein (LT) for origin recognition and helicase function, and otherwise recruits cellular DNA replication proteins to replicate its viral genomes. The primary critical interactions required for PyV DNA replication are the three interactions between LT and the cellular single-strand DNA binding complex (RPA) and DNA polymerase alpha- primase (Polprim), all three interactions are required for synthesis to be initiated. We have identified conditions both in living cells and in vitro where DDR prevents viral DNA replication, and shown that this is mediated by ATR, and correlates with phosphorylation of each of these three proteins, specifically: LT, the second subunit of RPA, and the second subunit of Polprim. Preliminary studies shown herein demonstrate that the phosphorylation of LT (evaluated by creating phosphomimetic mutations) doesn’t affect most functions of LT (DNA binding, hexamerization, ATPase, binding to RPA and Polprim, stimulation of polymerization by Polprim), but it does dramatically inhibit DNA helicase progression. Synthesis of primers is deficient in a second pathway independent of this DDR effect on LT. Naïve LT and Polprim with RPA purified from DDR-activated cells is severely inhibited for primer synthesis, suggesting that at the replication fork there is a coordinated inhibition of both leading and lagging strand synthesis, through helicase and priming suppression, respectively. The two Aims of this proposal are to prepare phosphomimetic mutations of both the RPA and Polprim complexes at their DDR sites, and evaluate the function of these two complexes as we have done with the phosphomimetic LT. Their functions alone, as well as in conjunction with both wt and phosphomimetic mutations of both of the other complexes will elucidate the detailed mechanisms behind how replication fork progression can be inhibited in a coordinated fashion in response to DDR. This has ramifications on how DNA replication stress can be targeted for cancer treatment and for treatment of human PyV infections.