Together, these events trigger and amplify the DDR signal. This argues that chromatin context does impact the DDR.It has also been speculated that the association of MDC with gHAX could counteract either dephosphorylation or chromatin remodeling events that would remove gHAX at breaks.BRCA, for which there are no homologues in yeast, has been shown to be part of at least three distinct complexes, the composition of which is determined by the binding of mutually exclusive adaptor proteins to the BRCT domain of BRCA.Abraxas, RAP and BRCC, like BRCA, all accumulate into IRIF, indicating that a functional BRCAA complex is recruited to DNA lesions.In cells depleted for either RAP or BRCC, the accumulation of the remaining subunits of the BRCAA complex into IRIF was impaired. The ultimate ubiquitin ligase is called an E ubiquitin ligase, which cooperates with an E ubiquitinactivating enzyme and an E ubiquitinconjugating enzyme.In yeast, all ubiquitin ligases share one E enzyme, which uses ATP to activate ubiquitin and subsequently transfers it for conjugation to an E enzyme.E enzymes interact with specific E ligases, which are often multiprotein complexes.E ligases are responsible for targeting ubiquitin from an E enzyme to a specific substrate.Polyubiquitylation usually targets proteins for proteasomemediated degradation.On their own, these domains can associate with these foci. This implicated ubiquitin chains in the accumulation of downstream DDR factors at sites of repair.Ubiquitin can be linked to itself through one of its seven lysine residues forming polymers in vivo. Because BRCA recruitment required MDC, these data coherently supported a model in which gHAXMDC binding led to ubiquitin conjugation at sites of DNA damage, which in turn was necessary for attracting and retaining the BRCAA complex and downstream DDR proteins. It accumulates at sites of DNA damage concomitantly with gHAX, MDC and NBS.As for many other DDR factors, RNF accumulation was lost in cells depleted for MDC. At the same time, it was found that MDC becomes phosphorylated in response to ionizing radiation on sites that are clearly distinct from the SDT CK targets.Indeed, the MDCN terminus contains four ATM andor ATR target sites, which, in a phosphorylated form, could serve as docking sites for the FHA domain of RNF. Subsequently, threoninetoalanine substitutions conrmed these points: the motifs within MDC are true target sites for the ATM kinase and, as such, are recognized both in vitro and in vivo by the RNF FHA domain.Thus, RNF functions downstream of gHAX and MDC in the DDR, yet accumulates in IRIF with kinetics that are comparable to NBS or MDC and faster than BP or BRCA.First, a knockdown of RNF abrogated the accumulation of both BP and BRCA. MG causes rapid depletion of nuclear ubiquitin by enabling nondegraded polyubiquitylated proteins to accumulate in the cytoplasm.In both mutants, there was neither accumulation of conjugated ubiquitin, nor accumulation of the factors dependent on its presence, namely BRCA, RAP and BP.However, the RNF mutations did not affect the phosphorylation and formation of gHAX foci.Collectively, these results conrmed that the E ligase activity of RNF mediates the accumulation of DDR factors that are downstream of the gHAXMDC interaction.