Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair.
ERCC1-XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5' to 3' away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1-XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1-XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.
Gaillard
Pierre-Henri L
PHL
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Wood
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002457:Cell Extracts; D017124:Conserved Sequence / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D004720:Endonucleases; D004926:Escherichia coli; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; C095888:xeroderma pigmentosum group F protein; D004247:DNA; C071447:ERCC1 protein, human; D004720:Endonucleases