Structural determinants for substrate binding and catalysis by the structure-specific endonuclease XPG.
XPG belongs to the Fen1 family of structure-specific nucleases and is responsible for the 3' endonucleolytic incision during mammalian nucleotide excision repair. In addition, it has ill-defined roles in the transcription-coupled repair of oxidative DNA damage and likely also in transcription that are independent of its nuclease activity. We have used DNA binding and footprinting assays with various substrates to gain insight into how XPG interacts with DNA. Ethylation interference footprinting revealed that XPG binds to its substrates through interaction with the phosphate backbone on one face of the helix, mainly to the double-stranded DNA. By comparing DNA binding and cleavage activity using single-/double-stranded DNA junction substrates differing in the length of the single-stranded regions, we have found that the 3' but not the 5' single-stranded arm was necessary for DNA binding and incision activity. Furthermore, we show that although a 5' overhang is not required for XPG activity, an overhang containing double-stranded DNA near the junction inhibits the nuclease but not substrate binding activity. Apparently, junction accessibility or flexibility is important for catalysis but not binding of XPG. These results show that XPG has distinct requirements for binding and cleaving DNA substrates.
Hohl
Marcel
M
Institute of Molecular Cancer Research, University of Zürich, August Forel Strasse 7, CH-8008 Zürich, Switzerland.
Thorel
Fabrizio
F
Clarkson
Stuart G
SG
Schärer
Orlando D
OD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D018983:DNA Footprinting; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D005090:Exodeoxyribonucleases / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000737:chemistry; D018384:Oxidative Stress; D011994:Recombinant Proteins; D018411:Spodoptera / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014157:Transcription Factors; D014162:Transfection; D000099250:DNA Excision Repair Protein ERCC-5
D004247:DNA; D004268:DNA-Binding Proteins; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C020133:exodeoxyribonuclease III