Does RAD52-driven MiDAS Antagonize 53BP1 in Human Cancer Cells
It is well known that in the cell cycle, DNA synthesis occurs in S phase and chromosome segregation occurs in M phase. However, a recently discovered phenomenon known as Mitotic DNA synthesis (MiDAS), provides evidence that not all DNA synthesis may occur in S phase (Minocherhomji et al., 2015). Under replication stress, MiDAS occurs at specific chromosome loci in both prophase and pro-metaphase cells, and if these late replication intermediates (LRI) are unresolved, non-disjunction of sister chromatids may occur in anaphase (Graber-Feesl, 2019). However, due to the highly error-prone nature of MiDAS, an appropriate resolution of LRIs is not always possible. As a result, MiDAS can also lead to chromosomal breakages as an exigency to avoid lethal chromosomal mis-segregation. These breakage sites are known to co-localize with focus formation of FANCD2 in normal mammalian and cancer cells (Graber-Feesl, 2019). Previous studies have also shown that another protein, RAD52, colocalizes with these sites in human cancer cells. RAD52-mediated SSA is implicated in MiDAS in cancer cells (Bhowmick, 2016). This activity of RAD52 is enhanced during the S/G2 phases of the cell cycle when 53BP1 is absent. 53BP1 is an important regulator of DSB repair, therefore, a loss of 53BP1 is hypothesized to cause excessive DNA end resection to enhance SSA and HBR and thus enhance MiDAS via the RAD52-driven mechanism (Bothmer, 2010). This was tested in U2OS cancer cells through two general procedures. First, it was tested to see if 53BP1 only inhibits MiDAS in human cancer cells and not in normal human cells. Depletion was confirmed via western blot and MiDAS immunohistostaining was used to mark early M-phase cells, points of DNA synthesis, and FANCD2 foci to show that 53BP1 does in fact only inhibit MiDAS in U2OS cancer cells. Then, it will be tested to see if RAD52 functions in the up-regulation of MiDAS as a result of 53BP1 depletion in cancer cells. Data is still being collected on this account and needs to be analyzed. Additionally, future directions include determining if the depletion of FANCD2 has no impact on the MiDAS level in 53BP1-depleted U2OS cancer cells.