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

Isabelle Dunand-Sauthier,Marcel Hohl,Fabrizio Thorel,Pascale Jaquier-Gubler,Stuart G Clarkson,Orlando D Schärer

doi:10.1074/jbc.m412228200

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. 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.

Highlights

Nucleotide excision repair (NER)1 is a remarkably flexible DNA repair pathway with the ability to eliminate a plethora of diverse DNA lesions caused by numerous environmental agents [1, 2]
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 nucleotide excision repair (NER) activity in vitro and in vivo
We made a deletion of a short sequence termed the D1 box, a stretch of 27 amino acids in the Nterminal part of the spacer region that is conserved in higher eukaryotes [51]

Summary

EXPERIMENTAL PROCEDURES

Construction of XPG Spacer Deletion Mutants—Deletion mutants were generated by digestion of pFastBac1-XPG [41] with HindIII and NdeI (⌬111–550), with NdeI and AseI (⌬554 –730), or with HindIII and AseI (⌬111–730), and the resulting pFastBac1-XPG fragments were ligated with a double-stranded adaptor DNA having corresponding sticky ends. DNA cleavage and binding assays were performed as described previously. Cell Culture Conditions and Preparation of Whole Cell Extracts—For the generation of whole cell extracts, SV40-transformed fibroblast cells XPCS1RO [43] were cultured in modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum and 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin at 37 °C in the presence of 5% CO2. Primary fibroblasts from a severely affected XP-G/CS patient (XP20BE) [45, 46] were cultured in modified Eagle’s medium supplemented with 15% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin and kept in a 5% CO2 humidified incubator.

Functional Characterization of the XPG Spacer Region

RESULTS

DISCUSSION