Ubiquitin regulates dissociation of DNA repair factors from chromatin

Involvement of the ubiquitin system in DNA repair and DNA damage signaling has been extensively studied during the last decade. Dynamic modification of proteins with ubiquitin after DNA damage has been shown to play important roles in virtually all DNA repair pathways [1]. In this regard, protein ubiquitylation has most frequently been associated with the recruitment of DNA repair factors – many containing ubiquitin-binding domains – to DNA lesions and/or flanking chromatin. A prominent example of this is monoubiquitylation of the replication clamp PCNA by the ubiquitin ligase RAD18 that leads to recruitment of error-prone DNA polymerases and ensuing translesion DNA synthesis. In addition, ubiquitylation of histone variant H2AX after DNA damage by the ubiquitin ligases RNF8 and RNF168 promotes the repair and signaling of DNA double strand breaks (DSBs) and leads to recruitment of downstream repair factors such as BRCA1 and 53BP1 [1]. Ubiquitin-dependent extraction of chromatin-associated proteins from repair sites is also now emerging as an important mechanism for maintaining chromatin and genome integrity. In this regard, we have recently shown that the Ku heterodimer, which mediates DSB repair by non-homologous end joining (NHEJ), is extracted from chromatin in an ubiquitin-dependent process [2]. NHEJ is the predominant DSB repair pathway in human cells, and is initiated by the binding of Ku to DNA ends. Ku forms a stable ring structure encircling DNA, and together with factors such as DNA-PKcs and PAXX, forms an assembly that mediates DSB repair by the DNA ligase IV/XRCC4/ XLF complex. Although components of the NHEJ pathway have been well characterized, the mechanisms that promote their dissociation from repair sites remain poorly understood. It has long been recognized that this dissociation is important because prolonged binding of Ku to chromatin after DSB repair would interfere with transcription and replication, thereby posing serious threats to the cell. We have found that the ubiquitin-like protein NEDD8 is recruited to DSB sites in human cells together with various components of the NEDD8-conjugation machinery [2]. Moreover, we established that NEDD8 conjugation actively occurs at such sites, and that depleting neddylation components impairs cell survival after DSB induction. Strikingly, through using NEDD8-activating enzyme inhibitor MLN4924, we established that neddylation does not appear to markedly affect DSB repair but rather promotes the removal of Ku and other NHEJ components from chromatin after repair has taken place. Previous work has established that a key role for neddylation is to activate a …