Abstract A04: Phosphorylation and ubiquitylation on the RPA-ssDNA platform promote homologous recombination

Replication stress is an important source of genome instability in cancer cells. Impediments to replication fork progression stimulate the accumulation of RPA-coated single-stranded DNA (RPA-ssDNA). RPA-ssDNA then recruits and activates a large number of genome maintenance factors including the master checkpoint kinase ATR to protect genomic integrity. Amongst the factors recruited onto RPA-ssDNA, we have previously shown that the E3 ubiquitin ligase PRP19, in addition to its central role as an RNA splicing factor, plays an integral part in the elicitation of the DDR during replication stress. Specifically, the ubiquitin ligase activity of PRP19 on the RPA-ssDNA platform is critical for the activation of the ATR checkpoint kinase and promotes replication fork stability during stress. How exactly is PRP19 tethered to RPA-ssDNA in response to damage and whether its ubiquitin ligase activity on this platform is important for specific DNA repair pathways remains unexplored.Here, we show that RPA ubiquitylation is potently triggered by genotoxic agents which target replication forks. The ubiquitylation of RPA and its interaction with PRP19 both correlate with RPA32 phosphorylation. In fact, we show that ubiquitylated RPA is also phosphorylated. Interestingly, a non-phosphorylatable RPA32 mutant interacts poorly with PRP19 and is not efficiently ubiquitylated. Furthermore, we identify a positively charged pocket on the PRP19 WD40 domain which interacts with RPA during replication s...
Source: Molecular Cancer Research - Category: Cancer & Oncology Authors: Tags: Homologous Recombination Defects: Poster Presentations - Proffered Abstracts Source Type: research