Common fragile sites (CFSs) are specific regions on the genome that are highly fragile and very sensitive to replication stress. Consequently they are hotspots for copy number variations (CNVs) and translocation. The genes located at CFSs share certain traits. They are extremely large and they replicate late in S phase. Transcription of the CFS genes can lead to unfinished or faulty replication, which triggers genomic instability. Replication problems that arise at CFSs have at least in part been attributed to transcription replication conflicts (TRCs).
Even though the consequences of TRCs on the CFS DNA are widely studied, little is known about the impact that they may have on the transcription of the CFS gene.
Here we use chicken DT40 cells to study the impact that replication stress and TRCs have on the CFS gene PARK2. PARK2 is an extremely large and transcriptionally active gene in DT40. The PARK2 gene has very large introns and it codes for an E3 ubiquitin ligase with a well-known role in mitophagy.
We find that mild replication inhibition compromises PARK2 expression, which results in reduced mitophagic activity. We see that the reduction in expression of PARK2 under replication stress conditions is FANCD2 dependent. Furthermore, we observed a 50% reduction in the fragility at the PARK2 CFS after reducing PARK2 size by approximately 12% by deleting part of intron 7. Finally, our preliminary results indicate that PARK2 transcription is exceptionally fast and it can be finished within one cell cycle despite its extremely large size.