Chromatin mobility contributes to the homology directed repair (HDR) of double strand breaks (DSBs) and replication stress. It has been shown that some DNA lesions specifically translocated to nuclear hubs of HDR activity. Centromeric integrity is key for proper chromosome segregation during cell division. Centromeres have unique chromatin and structural features that are essential for centromere maintenance. It has been recently shown that DSBs at centromeres recruit HDR machinery throughout the cell cycle by CENPA and H3k4me2 deposition. However, it remains unclear if human centromeric chromatin is mobilized during DSB repair, and if so, how this mobility is actuated.
Using CRISPR-Cas9 technology, we are investigating the dynamics and mobility of DSBs induced at centromeric alpha satellites. Preliminary data shows that DSBs induced in centromeric repeats have high mobility and cluster during the DNA damage response. ATR inhibition supresses centromeric break clustering, suggesting a possible resection-dependent mechanism underling mobility. By fusing the engineered ascorbate peroxidase (APEX2) to CENPA followed by mass spectrometry, we found that tubulin and mitotic assembly checkpoint proteins interact with CENPA during the DNA damage response. Interestingly, inhibition of tubulin polymerization by Nocodazole treatment supresses centromeric clustering. Overall, our overriding goal is to decipher the importance of the regulation of centromere mobility and integrity in the DNA damage response.