Characterization of a Putative Helix-Loop-Helix Motif in Nucleotide Excision Repair Endonuclease, XPG

Abstract

Complementation group G of xeroderma pigmentosum (XPG) is one of the most rare and pathophysiologically heterogeneous forms of this inherited disease. XPG patients exhibit varying phenotypes, from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological degeneracy and growth retardation of Cockayne's syndrome. The XPG gene encodes a 134-kDa nuclear protein that is essential for the incision steps of nucleotide excision repair. XPG protein contains a putative helixloop-helix (HLH) motif in the region that is most conserved among the members of structure-specific endonuclease family. To establish the functional significance of the HLH motif, we used several approaches, including theoretical modeling, functional complementation assay, structure-specific endonuclease assay, and DNA binding assay. A secondary structure of the motif was predicted by energy minimization and the Monte Carlo simulation and empirically proven using the circular dichroism to contain a high content of ␣-helix. When an XPG mutant lacking the HLH was overexpressed in UV135 cells, which have defects in the hamster homolog of XPG, the mutant gene failed to confer to the hamster cells the resistance to UV light. A recombinant XPG protein lacking the HLH motif was purified from insect cells and tested for a structure-specific endonuclease activity. The mutant protein failed to cleave the flap strand. A recombinant peptide containing the HLH (amino acids 758 -871) was expressed in and purified from bacteria, tested for DNA binding activity, and found to bind to a DNA substrate with the flap structure. These results suggest that the HLH motif is required for the catalytic and DNA binding activities of XPG.

Conserved Residues of Human XPG Protein Important for Nuclease Activity and Function in Nucleotide Excision Repair

“…7) may be relevant to its incision mechanism. Also relevant may be a helix-loop-helix motif found within the I region of XPG (64). Although no evidence was found for XPG multimers in the latter study, a recombinant peptide containing this helix-loop-helix motif was able to dimerize (64).…”

“…Also relevant may be a helix-loop-helix motif found within the I region of XPG (64). Although no evidence was found for XPG multimers in the latter study, a recombinant peptide containing this helix-loop-helix motif was able to dimerize (64). In the present work, co-expressed fulllength XPG protein and XPG lacking the conserved N region were found together by immunoprecipitation.…”

The tetratricopeptide repeat: a structural motif mediating protein-protein interactions

“…Interestingly, there are other protein interaction motifs that resemble the TPR motif in secondary structure; the paired amphipathic helix (PAH) motif, identified in certain transcription factors, (12) and the helix-loop-helix (HLH) motif, identified in transcription factors (13) and endonucleases. (14) Das et al (11) showed that the spatial arrangement of the antiparallel ␣-helices of the 14-3-3 protein resembles the arrangement of the helices within the PP5 TPR domain. The similarity in the structure of these motifs may be an example of convergent evolution toward a fundamentally important structure for protein interaction.…”

TPR Proteins in Plant Hormone Signaling

“…Three-dimensional structure data have shown that a TPR motif contains two antiparallel α-helices such that tandem arrays of TPR motifs generate a right-handed helical structure with an amphipathic channel that might accommodate the complementary region of a target protein. 4 Interestingly, there are other interaction domains that resemble the TPR motif in secondary structure; the paired amphipathic helix (PAH) motif and helix-loop-helix (HLH) motif, identified in certain transcription factors and endonucleases [6][7][8] and the antiparallel α-helices of 14-3-3 proteins. 9 The similarity in structure between these motifs most likely reflects a case of convergent evolution toward essential domains for protein interaction, which in turn may explain the abundance and functional importance of TPRs in nature.…”