LY2940680 vation and a DNA PK inhibitor did not block

53BP1vation, and a DNA PK inhibitor did not block 53BP1 targeting to damage sites. Our findings are therefore not congruent with a report that 53BP1 is required for CPT induced RPA2 hyperphosphorylation. It is also possible that ubiquitination itself, but not protein degradation, is LY2940680 required for DNAPK activation. Xu et al. have reported that proteasome inhibition causes the starvation of free Ub leading to defects in regulatory ubiquitination of histones H2A and H2B. γH2AX ubiquitination was suppressed by MG 132 in response to either CPT or NCS. This suggests that Ub starvation is responsible for the suppression of γH2AX ubiquitination in MG 132 treated cells and that DNA PK can be activated in the absence of Ub in response to NCS.
Furthermore, knockdown of RNF8, the responsible enzyme for H2AX ubiquitination, did not block CPT induced DNA PKcs autophosphorylation. Combined these data reveal that γH2AX ubiquitination is not required for DNA PK activation. Instead, the ubiquitination of other chromatin associated proteins may facilitate access of DNA PK to CPT induced DNA damage. Recently, Lin et al. have published that proteasome inhibition suppresses the generation of DSBs in response to CPT. In contrast to their data, MG 132 did not significantly decrease the percentage of γH2AX positive cells in our hands, which was suppressed by HU, suggesting that replication dependent DSBs are generated even in the presence of MG 132.
In addition, the effect of MG 132 on ATM and Chk1 phosphorylation was partial as shown by Lin et al, whereas MG 132 sharply abolished DNA PKcs and RPA2 phosphorylation, suggesting that MG 132 effect on DNA PK activation is definitely different from the effect on ATM/ATR activation. It has been reported that co treatment with proteasome inhibitor improves the effect of CPT against a colorectal cancer cell line. This synergistic effect of proteasome inhibition has been thought to be caused by the suppression of NF κB activation. CPT activates NF κB through the proteasome dependent degradation of I Ba which is inhibitory factor of NF κB. Given that proteasome inhibition blocks several steps in DNA damage signaling, including 53BP1 recruitment and DNA PK activation, it is plausible that a DNA repair defect may contribute to enhanced cytotoxicity of TopI poisons.
In higher eukaryotes ionizing radiation induced DNA double strand breaks are primarily repaired by the non homologous end joining pathway. Ku, a heterodimeric protein with a unique bridge and pillar structure has a very high affinity for DNA termini and binds to the site of the DSB. The DNA dependent protein kinase catalytic subunit is then recruited to the site of the break interacting with both the DNA terminus and the Ku heterodimer. The resulting heterotrimeric complex, termed DNA PK, is active as a serine/threonine protein kinase and can phosphorylate downstream substrates. As IR induced DSBs often contain other DNA structural damage including thymine glycols, ring fragmentation, 3, phosphoglycolates, 5, hydroxyl groups and abasic sites, processing of DNA termini is often necessary before ligation of the double strand break by the XRCC4/Ligase IV/ XLF complex can occur. A variety of enzymes have been implicated in DNA processing, including but not limited to, FEN 1, LY2940680 chemical structure.

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