Uncovering the impact of oxidative stress on centromere integrity

PROJECT SUMMARY The centromere is a key region of the chromosome that mediates kinetochore assembly and spindle attachment, thus ensuring proper chromosomal segregation. Compromised centromere integrity results in increased risk of chromosomal rearrangements and mis-segregation, which can lead to chromosomal instability (CIN) and large- scale genetic changes, characteristics commonly observed in cancer cells. One of the major threats to DNA integrity and genome stability is oxidative damage arising from the exposure to reactive oxygen species (ROS), known as oxidative stress (OS). Chronic exposure to OS is associated with centromeric dysfunction and increased centromere instability, yet the impact of OS specifically at the centromere is unclear. To investigate the DNA damage response at the centromere, our lab is using a specialized chemoptogenetic system designed to generate the common OS induced lesion 8-oxoguanine locally at centromeric DNA. This lesion occurs hundreds of times per day in a cell and is repaired through the Base Excision Repair (BER) pathway. My preliminary studies revealed immediate BER factor recruitment as well as shifts in the cell cycle and a large decrease in active DNA synthesis following OS at the centromere. In addition, I observed formation of DNA breaks and an increase in apoptosis 24h after treatment. Finally, RNA sequencing revealed changes in genes mapping to replication stress signaling and DNA damage repair. Based off these preliminary findings, I hypothesize that OS at the centromere leads to replication stress and increased CIN. In Aim 1, I will investigate the DNA damage response at the centromere and the contribution of DNA repair factors in maintenance of centromere integrity. In Aim 2, I will assess the impact of centromeric damage on chromosome segregation and DNA replication dynamics. These experiments will uncover the factors involved in mitigating the impact of OS and maintaining integrity at centromeres. The results will provide insight into how centromere dysfunction and subsequent CIN arise, helping further our knowledge of CIN associated cancers and informing potential therapeutic targets.