Abstract
RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2.
Highlights
Mammalian cells require the E3 ubiquitin ligase RAD18 for survival after the induction of various types of DNA damage
The results suggest that none of these sites is required for RAD18 ubiquitylation (Figure S1E), indicating the presence of another unknown putative ubiquitylation site in RAD18 that is required for mono-ubiquitylation in vivo
RAD18 accumulation at double-strand break (DSB) depends on RPA Having established that the Zinc finger domain of RAD18 is required for the formation of RAD18 foci throughout the cell cycle and upon irradiation, we investigated which proteins are required for RAD18 recruitment to chromatin
Summary
Mammalian cells require the E3 ubiquitin ligase RAD18 for survival after the induction of various types of DNA damage. The first step in the RDB pathway involves mono-ubiquitylation of PCNA by the RAD18-HR6A/B complex [8]. Mono-ubiquitylation of PCNA by the RAD18-HR6A/B complex recruits specific translesion synthesis polymerases that can incorporate nucleotides in the strand opposite the site of the DNA lesions [10]. RAD18 contains a RING finger that has been shown to be required for ubiquitylation of PCNA [1,10]. In addition to this domain, HR6A/B interacting domains [11,12,13], and a so-called SAP domain that shows binding affinity to single-stranded DNA (ssDNA) in vitro [14] have been identified. It was recently described that RAD18 contains a Zinc finger that functions as an ubiquitin binding domain [14,15,16,17]
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