Critical roles of ring finger protein RNF8 in replication stress responses

J Biol Chem. 2011 Jun 24;286(25):22355-61. doi: 10.1074/jbc.M111.232041. Epub 2011 May 10.


Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Survival / drug effects
  • Checkpoint Kinase 1
  • DNA Damage*
  • DNA Replication / drug effects
  • DNA Replication / genetics*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / metabolism*
  • G2 Phase / drug effects
  • G2 Phase / genetics
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Hydroxyurea / pharmacology
  • Protein Kinases / metabolism
  • Protein Transport / drug effects
  • Rad51 Recombinase / metabolism
  • Ubiquitin-Protein Ligases
  • Ubiquitination / drug effects


  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Histones
  • RNF8 protein, human
  • Ubiquitin-Protein Ligases
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Rad51 Recombinase
  • Hydroxyurea