Replicating Cells are Vulnerable to Mutations Induced by APOBEC3 Enzymes

Project Goal:

We are investigating the impact of the APOBEC3 cytidine deaminase enzymes on cellular genome integrity. APOBEC3 enzymes have recently been associated with several types of cancers and we find their mutational signature in pediatric leukemia genomes. Our hypothesis is that cellular DNA is susceptible to APOBEC3-induced mutation when single-stranded DNA (ssDNA) is exposed, since APOBEC3 enzymes are known to act primarily on ssDNA. Active investigations in the lab are seeking to determine specifically whether APOBEC3 activity is increased in cells undergoing replication or double-stranded break repair, two events during which ssDNA is present. We are using a novel technique known is iPOND (Isolation of Proteins on Nascent DNA) to label newly synthesized DNA at replication forks and precipitate it along with associated proteins for evaluation. Proteins associated with replicating DNA can be evaluated by immunoblotting. Various treatments will be used to induce replication stress in cells and impact of this stress on the association of APOBEC3 proteins with replicating DNA will be evaluated. This project has the potential to define new factors associated with the development of genome instability in pediatric leukemia, and may provide pathways for novel diagnostic and therapeutic options.