title: Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage.
abstract: DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8(-/-) mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: Sufang
authors.initials: S
authors.lastname: Zhang
authors.forename: Yajing
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ali
authors.initials: A
authors.lastname: Sarkeshik
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Thomson
authors.forename: Zhongtao
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ernest Y C
authors.initials: EY
authors.lastname: Lee
authors.forename: Marietta Y W T
authors.initials: MY
authors.lastname: Lee
mesh_terms: D000818:Animals; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D025821:Polyubiquitin / Q000378:metabolism; D021381:Protein Transport / Q000528:radiation effects; D059748:Proteolysis / Q000528:radiation effects; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000528:radiation effects; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000302:isolation & purification / Q000378:metabolism*; D054875:Ubiquitination / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; C486521:RNF8 protein, human; D011993:Recombinant Fusion Proteins; D025821:Polyubiquitin; C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C549817:Rnf8 protein, mouse; D044767:Ubiquitin-Protein Ligases; C521101:POLD4 protein, human; D004258:DNA Polymerase III; C579269:POLD4 protein, mouse