The Spacer Region of XPG Mediates Recruitment to Nucleotide Excision Repair Complexes and Determines Substrate Specificity

Abstract

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N-and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184 -210 and 554 -730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPG⌬184 -210 and XPG⌬554 -730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.

Suppression of UV-induced apoptosis by the human DNA repair protein XPG

“…39 The primary fibroblasts used were AG08803 and 96RD29 (from XP-G/CS patients XP20BE and XPCS2RO, respectively), XP125LO and GM01638 (from XP-G patients XP125LO and XP65BE, respectively), GM01630 (XP-A), GM00671 (XP-C), GM02965 (CS-A), GM01428 (CS-B) and 250BR (wild type). All experiments were carried out with primary fibroblasts at passage number o20.…”

“…39 They were stained with annexinV-FITC and PI (Apoptosis detection kit FITC, Alexis Biochemicals) following the manufacturer's instructions and analysed on a FACScan (Becton Dickinson).…”

“…39 The vector was pWIR8 that contains mouse CD8 cDNA as selectable marker. Details of lentiviral vectors and protocols can be found on http://www.tronolab.…”

“…Immunofluorescence was carried out as described 39 but with mouse monoclonal antibodies to Mdm2 (2A10, Oncogene) at 1 mg/ml, and to XPG (8H7, gift from RD Wood) at 1 : 100. Detection was performed with the anti-mouse IgG (H þ L) Alexat 488 (green) or 568 (red) conjugates (Molecular Probes) at 1 : 400.…”

XPG: Its Products and Biological Roles

“…The interaction of the N-terminal part of the spacer region with TFIIH has been shown to be important for the NER reaction. [24][25][26] An interaction with RPA has also been reported, but the functional significance of this interaction remains to be established. 27 The 300 amino acids at the Cterminal end of the protein, beyond the I region, are also engaged in protein-protein interactions; this region contains interaction sites with TFIIH and CSB and a PIP-box motif that mediates an interaction with PCNA.…”