USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Cell Cycle. 2014;13(1):106-14. doi: 10.4161/cc.26814. Epub 2013 Oct 24.


Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.

Keywords: 53BP1; BRCA1; DNA repair; RNF168; USP3; deubiquitinating enzyme; histone modification; ubiquitin ligase; γH2AX.

Publication types

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

MeSH terms

  • Cell Line
  • DNA Damage / genetics
  • DNA Repair / genetics
  • Histones / genetics*
  • Histones / metabolism
  • Humans
  • Lysine / genetics*
  • Protein Processing, Post-Translational
  • Signal Transduction
  • Ubiquitin-Protein Ligases / genetics*
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Specific Proteases / genetics
  • Ubiquitin-Specific Proteases / metabolism*
  • Ubiquitination / genetics


  • H2AX protein, human
  • Histones
  • RNF168 protein, human
  • Ubiquitin-Protein Ligases
  • USP3 protein, human
  • Ubiquitin-Specific Proteases
  • Lysine