RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

Abstract

dEfficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1␣ (HP1␣). Our study indicates that RAD6 physically interacts with HP1␣ and ubiquitinates HP1␣ at residue K154, thereby promoting HP1␣ degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1␣ exhibits an inverse relationship and correlates with the survival rate of patients. Double-strand breaks (DSBs) in DNA are considered the most deleterious types of DNA damage and pose a great threat to the integrity of the genome. Two pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), have evolved in mammals to repair the broken ends that characterize DSBs (1). The HR pathway is a precise repair pathway, wherein missing and damaged sequence information is copied from sister chromatids to catalyze the repair (2, 3). In contrast, the repair of DNA DSBs by NHEJ is more error prone and often leads to insertions, deletions, or other types of chromosomal rearrangements. The accumulation of DNA mutations, due to either unrepaired broken ends or improper repair, is thought to increase the incidence rate of cancer and other types of diseases (4, 5).Mounting evidence indicates that the ubiquitination of DSB repair proteins plays an important role in regulating DSB repair in mammals (6-8). Ubiquitination is classified into two types, monoubiquitination and polyubiquitination, depending on the number of ubiquitin molecules that become posttranslationally attached to target proteins. Monoubiquitinated proteins have been shown to participate in nonproteolytic pathways such as receptor trafficking, signal transduction, gene transcription, and DNA repair, while the polyubiquitination of substrates often leads to protein degradation either through the 26S proteasome pathway or through the autophagy pathway (9-12).Ubiquitination is catalyzed by a series of enzymes that includes the ubiquitin activation enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin ligase (E3) (13). RAD6 is an E2 ubiquitin-conjugating enzyme with a well-described role in stimulating the repair of UV-induced DNA damage (7,14). In budding yeast, RAD6 interacts with RAD18 to catalyze the monoubiquitination of proliferating cell nuclear antigen (PCNA) on lysine 164, thereby promoting the error-prone DNA damage repair pathway by recruiting low-fidelity polymerases. Interestingly, the interaction between the RAD6-RAD18 complex and the Ubc13-MMS2-Rad5 complex facilita...

Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage

“…3A). These two reporter systems were described in our previous report (12). RAD6A overexpression promoted both HR and NHEJ repair efficiency, consistent with the findings of our previous work (12).…”

“…These two reporter systems were described in our previous report (12). RAD6A overexpression promoted both HR and NHEJ repair efficiency, consistent with the findings of our previous work (12). Moreover, RAD6A-promoted HR and NHEJ repair, especially HR repair, was abolished by proteasome inhibition (treatment with the proteasome-specific inhibitor MG132), indicating a novel role of proteasomes in the regulation of DNA damage repair (Fig.…”

“…2E). Intriguingly, most of these biological functions have been reported by others (9,12,15,(19)(20)(21)(22), further validating the data from our mass spectrometry analysis and the related assays. However, when cells were irradiated with X rays, there was an obvious functional shift of the interaction networks of the RAD6A interaction partners.…”

“…We found that RAD6 regulates the ubiquitination and degradation of HP1␣ in human cells and the degradation of HP1␣ by RAD6 results in relatively open chromatin conditions, further facilitating the DNA damage repair efficiency, especially the HR repair efficiency (12). Multiple pathways are likely involved in the RAD6-mediated DNA damage repair process.…”

Triggering of Autophagy by Baicalein in Response to Apoptosis after Spinal Cord Injury: Possible Involvement of the PI3K Activation

“…Interestingly, autophagy plays an important role in the repair of nerve function in the CNS [8][9][10]. Moreover, we found that levels of autophagy are significantly increased after SCI, and that neuronal autophagy is associated with neurological function recovery as well as inhibition of apoptosis in the spinal cord [11].…”