The non-catalytic function of XPG protein during dual incision in human nucleotide excision repair.
XPG is a member of the FEN-1 structure-specific endonuclease family. It has 3'-junction cutting activity on bubble substrates and makes the 3'-incision in the human dual incision (excision nuclease) repair system. To investigate the precise role of XPG in nucleotide excision repair, we mutagenized two amino acid residues thought to be involved in DNA binding and catalysis, overproduced the mutant proteins using a baculovirus/insect cell system, and purified and characterized the mutant proteins. The mutation D77A had a modest effect on junction cutting and excision activity and gave rise to uncoupled 5'-incision by mammalian cell-free extracts. The D812A mutation completely abolished the junction cutting and 3'-incision activities of XPG, but the excision nuclease reconstituted with XPG (D812A) carried out normal 5'-incision at the 23rd-24th phosphodiester bonds 5' to a (6-4) photoproduct without producing any 3'-incision. It is concluded that Asp-812 is an active site residue of XPG and that in addition to making the 3'-incision, the physical presence of XPG in the protein-DNA complex is required non-catalytically for subsequent 5'-incision by XPF-ERCC1.
Wakasugi
M
M
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.
Reardon
J T
JT
Sancar
A
A
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5
D004247:DNA; D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5