Coordinated regulation of XPA stability by ATR and HERC2 during nucleotide excision repair

Oncogene. 2014 Jan 2;33(1):19-25. doi: 10.1038/onc.2012.539. Epub 2012 Nov 26.

Abstract

ATR (ATM and Rad3-related) is an essential regulator of the nucleotide excision repair (NER) mechanism. For NER activation, ATR phosphorylates XPA, the rate-limiting factor in the NER pathway. However, the role of XPA phosphorylation at serine 196 by ATR has been elusive. Here we show that ATR-mediated XPA phosphorylation enhances XPA stability by inhibiting HERC2-mediated ubiquitination and subsequent degradation. We analyzed stabilization of XPA with substitutions of Ser 196 either to aspartate (S196D), a phosphomimetic mutation, or to alanine (S196A), a phosphodeficient mutation. Upon ultraviolet damage, ATR facilitated HERC2 dissociation from the XPA complex to induce XPA stabilization. However, this regulation was abrogated in S196A-complemented XPA-deficient cells due to persistent association of HERC2 with this XPA complex, resulting in enhanced ubiquitination of S196A. Conversely, the S196D substitution showed delayed degradation kinetics compared with the wild-type and less binding with HERC2, resulting in reduced ubiquitination of S196D. We also found that XPA phosphorylation enhanced the chromatin retention of XPA, the interaction with its binding partners following DNA damage. Taken together, our study presents a novel control mechanism in the NER pathway by regulating the steady-state level of XPA through posttranslational modifications by which ATR-mediated phosphorylation induces XPA stabilization by antagonizing HERC2-catalyzed XPA ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Cell Line, Tumor
  • DNA Repair*
  • Gene Expression
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Humans
  • Phosphorylation
  • Protein Stability
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ubiquitination
  • Xeroderma Pigmentosum Group A Protein / genetics
  • Xeroderma Pigmentosum Group A Protein / metabolism*

Substances

  • Guanine Nucleotide Exchange Factors
  • HERC2 protein, human
  • RNA, Messenger
  • XPA protein, human
  • Xeroderma Pigmentosum Group A Protein
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins