Inhibition of Proteasomal Degradation of Rpn4 Impairs Nonhomologous End-Joining Repair of DNA Double-Strand Breaks

Xie Y (2010) Inhibition of Proteasomal Degradation of Rpn4 Impairs Nonhomologous End-Joining Repair of DNA Double- Strand Breaks. PLoS ONE 5(4): e9877. doi:10.1371/journal.pone.0009877 Inhibition of Proteasomal Degradation of Rpn4 Impairs Nonhomologous End-Joining Repair of DNA Double-Strand Breaks Donghong Ju 0 Xiaogang Wang 0 Seung-Wook Ha 0 Jiejun Fu 0 Youming Xie 0 Maria G. Masucci, Karolinska Institutet, Sweden 0 1 Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America, 2 Department of Pathology, Wayne State University School of Medicine , Detroit, Michigan , United States of America Background: The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. Methodology/Principal Findings: Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. Conclusion/Significance: This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ. - Funding: This work was supported by National Science Foundation grant MCB-0816974 to YX. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The S. cerevisiae RPN4 gene (also named SON1 and UFD5) was originally isolated as a suppressor of sec63-101, a temperaturesensitive mutant of SEC63, which encodes an essential component of the endoplasmic reticulum translocation channel [1,2]. Subsequent work showed that deletion of RPN4 inhibits the degradation of several model substrates of the N-end rule and UFD (Ub fusion degradation) pathways, suggesting the involvement of Rpn4 in proteasomal degradation [3]. The exact functional role of Rpn4 in protein degradation, however, remained unclear until recent studies revealed that Rpn4 is a transcription factor for the proteasome genes [4,5]. This finding explains why the proteasome activity is diminished in an rpn4D mutant. Interestingly, Rpn4 is an extremely short-lived protein (t1/2#2 min) and degraded by the proteasome [510]. Moreover, stabilization of Rpn4 by inhibition of the proteasome activity leads to an increase in the expression levels of the proteasome genes [11,12]. Together, these observations led to a model in which the proteasome homeostasis is regulated by a negative feedback circuit. On the one hand, Rpn4 upregulates the proteasome genes; on the other hand, Rpn4 is rapidly degraded by the assembled/ active proteasome. The Rpn4-proteasome negative feedback circuit provides an efficient and sensitive means to control the in vivo proteasome abundance. The proteasome genes in higher eukaryotes including humans are regulated by a similar negative feedback mechanism even though the homologs of Rpn4 have not yet been identified [1316]. In addition to the proteasome genes, Rpn4 appears to influence the expression of a large number of other genes involved in protein ubiquitylation, DNA repair and other cellular processes [4,1723]. Interestingly, the promoter of RPN4 carries the binding sites for heat-shock transcription factor (Hsf1), multidrug resistance-related transcription factors (Pdr1 and Pdr3), and Yap1, a transcription factor that plays an important role in response to oxidation and DNA damage [18,24,25]. These transcription factors are activated by a variety of environmental stressors and in turn induce RPN4 expression [11,12,17,18,2426]. These observations suggest that Rpn4 may serve as a major stress- responsive mediator. The Rpn4-proteasome negative feedback loop likely plays a central role in the Rpn4-mediated stress response network, not only by maintaining the proteasome homeostasis but also by gauging the expression levels of other Rpn4 target genes through proteasomal degradation of Rpn4. In support of this hypothesis, our recent studies demonstrated that disruption of each of the two branches of the Rpn4-proteasome negative feedback loop, namely Rpn4induced proteasome expression and proteasomal degradation of Rpn4, severely reduces cell viability under stressed conditions [27,28]. Rpn4 can be degraded by two distinct mechanisms, ubiquitin (Ub)-dependent and -independent [6]. Our recent studies showed that the N-terminal 10 amino acids are required for the Ubindependent degradation of Rpn4, whereas residues 211229 constitute the Ub-dependent degradation signal [610]. Simultaneous deletions of residues 110 and 211229 substantially stabilize Rpn4, and yet, do not impair its transcriptional activity [28,29]. Taking advantage of this stabilized Rpn4 mutant (Rpn4D110/D211229, referred to as Rpn4* for abbreviation), we demonstrated that inhibition of Rpn4 degradation causes cell hypersensitivity to DNA damage, particularly in response to high doses of DNA damaging agents [28]. It is possible that expression of Rpn4* may affect checkpoint activation in response to DNA damage. Alternatively, it may lead to a defect in DNA repair. In this study we sought to understand how inhibition of Rpn4 degradation sensitizes cells to DNA damage. We found that expression of Rpn4*, while imposing no effect on DNA checkpoint activation, displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. We further demonstrated that expression of Rpn4* impairs NHEJ but not homologous recombination (HR) repair of DSBs. The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced in the cells expressing Rpn4*. Interestingly, Rpn4 is recruited to a DSB and inhibition of Rpn4 degradation may cause a concomitant delay of the dissociation of Rpn4 and the proteasome from the DSB. These observations suggest that inhibition of Rpn4 degradation may affect NHEJ through different mechanisms. Inh (...truncated)