Inhibitors of the Proteasome Suppress Homologous DNA Recombination in Mammalian Cells

Abstract

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA doublestrand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, ;H2AX, or Mre11. These data show that the proteasomemediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways. [Cancer Res 2007;67(18):8536-43]

RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair

“…Proteasome function has been linked to the DDR previously by studies investigating the effects of proteasome inhibitors on IRIF and DNA repair by HR (Jacquemont and Taniguchi 2007;Murakawa et al 2007). Furthermore, a previous study reported enhanced ubiquitylation of MDC1 on chromatin following IR, linking this to proteasome-mediated disassembly of MDC1 and gH2AX foci, although the ubiquitin E3 ligases remained unknown (Shi et al 2008).…”

“…While the ubiquitin-proteasome system (UPS) was originally described as the main protein turnover/ degradation machinery of eukaryotic cells, it is now evident that it is also a major nondegradative regulator of various cellular processes, including the DDR (Kirkin and Dikic 2007;Bergink and Jentsch 2009;Motegi et al 2009;Al-Hakim et al 2010). Furthermore, it has been shown that proteasomes accumulate on damaged chromatin, suggesting that they may promote DDR signaling as well as DDR-dependent protein turnover and HR (Ustrell et al 2002;Blickwedehl et al 2007;Jacquemont and Taniguchi 2007;Murakawa et al 2007;Shi et al 2008;Motegi et al 2009;Al-Hakim et al 2010;BenAroya et al 2010;Levy-Barda et al 2011). The kinetics and mechanisms governing proteasome accumulation at DNA damage regions and the functions and targets of proteasomes at such sites, however, remain largely obscure.…”

The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

“…2 A and B), indicating that each is simultaneously present at many of the same DSB regions throughout much of the DNA damage response. Both ␥H2AX ubiquitination and DSB targeting of several repair proteins are strongly reduced at 30 min following administration of the proteasome inhibitor MG132 (7,8,26,27). Proteasome inhibition reduces nuclear ubiquitin concentrations by preventing degradation of poly-ubiquitinated proteins in the cytosol and regeneration of free ubiquitin pools.…”

Unrepairable DNA double-strand breaks that are generated by ionising radiation determine the cell fate of normal human cells

“…Polyubiquitylation is maintained at high levels in unrepairable IRIF (Fig. 1C), and pretreatment of cells with the polyubiquitylation inhibitor MG132 (Mailand et al, 2007;Murakawa et al, 2007;Shao et al, 2009) results in the formation of IRIF that lacks 53BP1 immediately after irradiation (Fig. 4A).…”