title: Human RAD18 interacts with ubiquitylated chromatin components and facilitates RAD9 recruitment to DNA double strand breaks.
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.
authors.affiliation: Department of Reproduction and Development, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Akiko
authors.initials: A
authors.lastname: Inagaki
authors.forename: Esther
authors.initials: E
authors.lastname: Sleddens-Linkels
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.forename: Richard G
authors.initials: RG
authors.lastname: Hibbert
authors.forename: Titia K
authors.initials: TK
authors.lastname: Sixma
authors.forename: Jan H J
authors.initials: JH
authors.lastname: Hoeijmakers
authors.forename: J Anton
authors.initials: JA
authors.lastname: Grootegoed
authors.forename: Willy M
authors.initials: WM
authors.lastname: Baarends
mesh_terms: D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D014466:Ultraviolet Rays; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D008164:Luminescent Proteins; C485954:RAD18 protein, human; D025801:Ubiquitin; C069930:rad9 protein; D044767:Ubiquitin-Protein Ligases