Indirect evidence suggesting that ATR promotes HR include the findings that ATR phosphorylates a number of substrates known to directly affect HR. Additionally, the PIKK inhibitor caffeine, which inhibits ATR kinase activity as well as other PIKKs such as ATM and DNA-PK, decreased site directed DSB-induced HR. Further, reduced levels of ATR rendered cells sensitive to PARP1 inhibition, a phenomenon often associated with a HR defect. More direct studies addressing the role of ATR in HR have also been Reversine conducted. Following expression of a kinase dead ATR mutant protein, Wang et al., found that HR frequency decreased following a restriction enzyme-mediated site-directed DSB, presumably in a dominant-negative fashion. This would suggest that ATR promotes HR following DNA damage. In contrast, Chanoux et al. found that the conditional deletion of ATR in mouse embryonic fibroblasts resulted in an approximate two-fold increase in spontaneous-induced RAD51 foci, and that this result was further increased in the presence of the replication stress inducing agent aphidicolin. However, it should be noted that though RAD51 protein is necessary in an early step of HR, its accumulation in nuclear foci does not necessarily indicate completion of the HR repair event. RPE HR reversion LY2109761 events were scored by the phenotypic appearance of a cell with their cytoplasm packed with brown melanosomes. Unless otherwise noted, the number of pigmented cells or groups of pigmented cells are counted for the entire RPE whole mount by using a stereomicroscope with criteria for what constitutes a single eye spot set forth by Bishop et al. To define Cre activity in our system we used a nuclear localized b-galactosidase Cre reporter system. The nuclear localization of this enzymatic reporter is paramount to our system because it allows for the detection of both Cre activity and HR in the same cell. For each eye spot counted, the presence of a bgalactosidase positive stained nucleus was also recorded. Therefore, the frequency of eye spots per RPE with or without bgalactosidase staining was used to calculate the frequency of HR. Additionally, the overall percentage of b-galactosidase staining for each RPE whole mount was visually assessed and assigned a percentage. Genomic instability is known to be a major factor in cancer development, thus understanding the systems that influence genomic stability in the normal somatic tissue is key to understanding cancer predisposition. Homologous recombination is a key DNA repair pathway that is usually considered to have high fidelity. However, either too much or too little HR can be deleterious, resulting in genomic alterations that can promote cancer -Notoginsenoside-R2.png)
development. ATR is believed to be the primary signaling kinase responding to replication stress in order to prevent deleterious lesions that might be caused by replication fork stalling. Previous studies have found that ATR deficient cells have increased levels of the DSB marker H2AX following exposure to agents that induce replication stress. Therefore, we suggest that ATR reduces the incidence of DSBs that can occur as a result of replication fork collapse by promoting HR at a stalled replication fork. In apparent contrast to this model, an increase in both spontaneous and replication damageinduced RAD51 foci in the absence of ATR has been reported.