AOH1996, a small molecule ligand of PCNA, modulates PCNA interaction with RPB1

Targeting transcription replication conflicts, a major source of endogenous DNA double stranded breaks and genomic instability, could have important therapeutic implications. When transcription and DNA replication machineries encounter each other on chromosomes, one way to resolve the conflict is temporarily dislodging RNA polymerase II from the conflict sites to enable the replication fork to proceed. Proliferating cell nuclear antigen (PCNA), an essential component of replisomes, plays a critical role in this process. We discovered a small molecule ligand (AOH1996) of PCNA, which targets a binding pocket adjacent to the outer surface region of PCNA known to interact with PIP box and APIM motif proteins. Orally administrable, AOH1996 suppresses tumor growth but causes no discernable side effects. In addition, we found that AOH1996 treatment can stabilize interaction between PCNA and RPB1, the largest subunit of RNA polymerase II. To determine whether the effect of AOH1996 is mediated through affecting PCNA interaction with RPB1, we mutated Y418 within the APIM motif of RPB1 to an alanine in the SK-N-AS cell line by CRISPR and compared changes in protein expression profiles caused by AOH1996 in the parent and mutant SK-N-AS cells.