DNA twice strand breaks are a particularly toxic form of DNA damage and the mammalian cell has evolved an intricate set of responses to repair this type of DNA lesion. or homeostatic mechanisms following completion of repair remain largely unexplored. Recent publications by our laboratories and the Medema laboratory shed new light on this issue. Both publications showed that this Wild-type p53-Induced Phosphatase 1 (WIP1) directly dephosphorylates γH2AX. WIP1 migrates to the sites of irradiation-induced foci (IRIF) though at a delayed rate relative to MDC1 and mediates γH2AX dephosphorylation presumably after DNA repair is usually total. This prevents recruitment of other repair factors such as MDC1 and 53BP1 to the DNA damage sites and promotes the dissolution MK-2048 of IRIF. In addition overexpression of WIP1 has a suppressive effect on DNA double strand break repair. Taken together MK-2048 these reports further implicate WIP1 as a critical homeostatic regulator of the DDR. null mice by the Donehower laboratory also resulted in enhanced levels of tissue γH2AX compared to wildtype mice. The effects of WIP1 on H2AX phosphorylation were not dependent on ATM as these effects were observed in ATM-deficient cells and mice. In cellular immunolocalization assays the Medema laboratory showed that WIP1 and γH2AX co-localized in the chromatin fractions of DNA damaged cells. Importantly they also exhibited that MDC1 and WIP1 co-migrated to laser-initiated DNA damage regions of cells though WIP1 migrated at a delayed rate compared to MDC1. MDC1 binds directly to γH2AX in damage foci so co-localization of WIP1 and γH2AX is usually assumed. Rabbit Polyclonal to FA13A (Cleaved-Gly39). Thus the role of WIP1 in damage foci (IRIF) formation was investigated. In WIP1 overexpressing cells γH2AX MDC1 or 53BP1 foci formation after exposure to IR is usually robustly downregulated whereas WIP1 depletion enhanced γH2AX foci formation and MDC1 and 53BP1 foci association. Finally the Donehower/Lu laboratories utilized two assays to examine rates of DSB repair. These assays revealed that reduction of WIP1 enhanced γH2AX association with individual DSBs and enhanced the rates at which DSBs were repaired. In another assay overexpression of WIP1 was shown to suppress homologous recombination (HR)- and non-homologous end joining (NHEJ)-mediated DSB repair while WIP1 knockdown by either siRNA or WIP1 inhibitors resulted in enhanced steps of DSB repair. The combined results by both groups provide a strong argument that WIP1 is usually a key γH2AX phosphatase that plays an important role in homeostatic regulation of DSB repair in response to DNA damage (Table 1). Because WIP1 likely targets many elements of the DDR pathway known (Chk1 Chk2 p53 Mdm2 Mdmx and ATM) and unknown we cannot attribute γH2AX dephosphorylation solely to WIP1 activity. Moreover the other aforementioned γH2AX phosphatases surely play a role as well. Rather WIP1 dephosphorylation of γH2AX is likely to be one of many contributing factors that negatively modulate the DDR pathways and enable cellular homeostasis after DSB repair is usually complete. Table 1 Major findings of Macurek et al. (2010) and Moon et al. (2010) The presence of multiple γH2AX kinases may give a clue as to why cells have multiple γH2AX phosphatases. MK-2048 Although all three PIKKs ATM ATR and DNA-PKcs phosphorylate H2AX they are activated by unique mechanisms and seem to have distinct target subsets of H2AX. Previous studies suggested that in response to DSB H2AX near the DSB is usually targeted by ATM and DNA-PKcs and that H2AX further away from the MK-2048 DSB is usually phosphorylated either by MK-2048 ATM in a MDC1-impartial manner or by ATR after DSB resection. Intriguingly Savic et al. suggested that γH2AX phosphorylation round the DSB can induce γH2AX phosphorylation even on other chromosomes surrounding the DSB.37 38 On the other hand in response to replication MK-2048 stress uncovered single-stranded DNA recruits ATR in cooperation with RPA and ATRIP to mediate γH2AX phosphorylation.39 We discovered that WIP1 impairs IR-induced γH2AX phosphorylation in both ATM proficient and deficient cells suggesting that WIP1 targets γH2AX generated by ATR and DNA-PKcs. Indeed WIP1 suppresses both HR- and NHEJ-mediated.