Abstract
Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structure-specific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328-355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg339) and XPG (Arg992) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.
Highlights
Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and [] photoproducts
We conclude that the C-terminal region from residues 364 to 380 of FEN-1 is not essential for proliferating cell nuclear antigen (PCNA) binding activity and makes little if any contribution to this activity
This contrasts with the previous report that truncated FEN-1 is unable to bind to PCNA in gel filtration and affinity bead pull-down assays [12]
Summary
24522–24529, 1997 Printed in U.S.A. The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21*. Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and [] photoproducts These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER).. We report domain mapping experiments to pinpoint the PCNA-binding region of FEN-1 and show that the small region responsible for activity is conserved in XPG. We identify a convergent evolutionary relationship between the PCNA-binding domains of the DNA damage-inducible inhibitor p21 and the repair endonuclease XPG and show that these domains compete for binding to PCNA
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