Characterization of a putative helix-loop-helix motif in nucleotide excision repair endonuclease, XPG.
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 helix-loop-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 alpha-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.
Park
M S
MS
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. park@telomere.lanl.gov
Valdez
J
J
Gurley
L
L
Kim
C Y
CY
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry*; D018257:Helix-Loop-Helix Motifs; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5
D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5