DNA damage response (DDR) maintains genome integrity through checkpoint activation and lesion repair. While ATM and ATR are essential in DDR, mechanisms regulating their activation remain unclear. BMI1 is a component of the polycomb repressive complex 1 (PRC1), and contributes to PRC1’s E3 ubiquitin (E3-Ub) ligase activity though binding the catalytic subunit RING2. BMI1 binds RING2 through its ring finger (RF) domain. The E3-Ub ligase activity contributes to BMI1-deirved facilitation of the homologous recombination-based repair of DNA double-stranded breaks (DSBs). My research demonstrates that BMI1 reduces ATM and ATR activation during DDR. DSBs and single-strand DNA (ssDNA) lesions respectively activate ATM and ATR. ATM subsequently phosphorylates CHK2 at threonine 68 (CHK2pT68) and induces G2/M arrest. ATR produces CHK1pS345 and S-phase arrest. Both kinases phosphorylate histone H2AX at serine 139 (γH2AX) to prepare for lesion repair. Hydroxyurea initiates DDR via producing ssDNA lesions, and increases ATR activation (phosphorylation of T1989/ATR pT1989), CHK1pS345, γH2AX, and S-phase arrest. These events were significantly reduced and enhanced following the respective BMI1 overexpression and BMI1 knockdown in MCF7 and DU145 cells. BMI1 also displays similar effects towards ATM during DDR induced by etoposide-caused DSBs. Activation of ATM and ATR requires the formation of the ATM-NBS1 and ATR-TOPBP1 complexes. We observed that BMI1 interacted with NBS1 or TOPBP1. Deletion of the RF domain from BMI1 did not affect the associations and also had no effects on BMI1’s activity in reducing ATM activation and ATR-mediated CHK1 pS345. Collectively, our research suggests that BMI1 attenuates ATM and ATR signaling independently of the E3-Ub ligase activity. Genotoxic treatments elicit DDR in cells that are directly exposed and also in cells that are not exposed, a phenomenon known as bystander effect (BE). However, it remains unclear what mediates the BE. Microvesicles are small membrane-enclosed sacks that are shed from donor cells and communicate specific messages to recipient cells. We demonstrated that microvesicles isolated from cells treated with etoposide and ultraviolet induced BE in recipient cells. Neutralization of microvesicles through annexin V reduced the microvesicles-associated BE. / Thesis / Doctor of Philosophy (PhD)

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and check-point activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.