Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase δ for degradation in response to DNA damage

J Biol Chem. 2013 Feb 1;288(5):2941-50. doi: 10.1074/jbc.M112.423392. Epub 2012 Dec 11.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • DNA Damage*
  • DNA Polymerase III / metabolism*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Half-Life
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Mice
  • Mice, Knockout
  • Models, Biological
  • Polyubiquitin / metabolism
  • Protein Transport / radiation effects
  • Proteolysis* / radiation effects
  • RNA, Small Interfering / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Subcellular Fractions / metabolism
  • Subcellular Fractions / radiation effects
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases / isolation & purification
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination / radiation effects
  • Ultraviolet Rays


  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • RNA, Small Interfering
  • RNF8 protein, human
  • Recombinant Fusion Proteins
  • Polyubiquitin
  • UBE2D3 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Rnf8 protein, mouse
  • Ubiquitin-Protein Ligases
  • POLD4 protein, human
  • DNA Polymerase III
  • POLD4 protein, mouse