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PMID:10413517

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Citation

McCutchen-Maloney, SL, Giannecchini, CA, Hwang, MH and Thelen, MP (1999) Domain mapping of the DNA binding, endonuclease, and ERCC1 binding properties of the human DNA repair protein XPF. Biochemistry 38:9417-25

Abstract

During nucleotide excision repair, one of the two incisions necessary for removal of a broad spectrum of DNA adducts is made by the human XPF/ERCC1 protein complex. To characterize the biochemical function of XPF, we have expressed and purified the independent 104 kDa recombinant XPF protein from E. coli and determined that it is an endonuclease and can bind DNA in the absence of the ERCC1 subunit. Endonuclease activity was also identified in a stable 70 kDa proteolysis fragment of XPF obtained during protein expression, indicating an N-terminal catalytic domain. Sequence homology and secondary structure predictions indicated a second functional domain at the C-terminus of XPF. To investigate the significance of the two predicted domains, a series of XPF deletion fragments spanning the entire protein were designed and examined for DNA binding, endonuclease activity, and ERCC1 subunit binding. Our results indicate that the N-terminal 378 amino acids of XPF are capable of binding and hydrolyzing DNA, while the C-terminal 214 residues are capable of binding specifically to ERCC1. We propose that the N-terminal domain of XPF contributes to the junction-specific endonuclease activity observed during DNA repair and recombination events. In addition, evidence presented here suggests that the C-terminal domain of XPF is responsible for XPF/ERCC1 complex formation. A working model for the XPF protein is presented illustrating the function of XPF in the nucleotide excision pathway and depicting the two functional domains interacting with DNA and ERCC1.

Links

PubMed Online version:10.1021/bi990591+

Keywords

Binding Sites; DNA/metabolism; DNA Repair; DNA-Binding Proteins/genetics; DNA-Binding Proteins/isolation & purification; DNA-Binding Proteins/metabolism; Endonucleases/metabolism; Escherichia coli/genetics; Humans; Peptide Fragments/biosynthesis; Peptide Fragments/genetics; Peptide Fragments/isolation & purification; Peptide Mapping; Protein Structure, Tertiary; Proteins/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:ERCC1

part_of

GO:0000110: nucleotide-excision repair factor 1 complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:XPF

involved_in

GO:0006289: nucleotide-excision repair

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:XPF

involved_in

GO:0033683: nucleotide-excision repair, DNA incision

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:XPF

enables

GO:0004520: endodeoxyribonuclease activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:XPF

enables

GO:0003697: single-stranded DNA binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:XPF

part_of

GO:0000110: nucleotide-excision repair factor 1 complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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