The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity.

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. XPGDelta184-210 and XPGDelta554-730 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.