Abstract
The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.
Highlights
Minus means that no incision products were detected
K179A) showed reduced RPA70 binding and fewer DNA incisions but had normal damaged DNA binding activity. These results indicate that the binding of XPA to RPA70 is important for NER and that amino acid substitutions in some XPA mutants result in both reduced damaged DNA binding and RPA70 binding activities
Analysis of Mutant XPA Proteins Defective in RPA Binding— we examined the mutant XPA proteins that were defective in both RPA32 and RPA70 binding but normal in damaged DNA binding activity
Summary
Preparation of Mutant XPA Proteins—To generate His6tagged XPA expression constructs, wild-type and N⌬29 XPA cDNA [14] were cloned in-frame in pET16b (Novagen). The wells were incubated with PBST (PBS/DTT, 0.1% Tween 20) containing 5% skim milk for 1 h and washed twice with PBST. The wells were incubated with anti-RPA32 monoclonal antibody [29] in PBST containing 1% skim milk for 1 h and washed five times with PBST. Mutant XPA (600 ng) was incubated with anti-XPA polyclonal antibody (1 g; Santa Cruz Biotechnology, FL-273) in NETN buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 0.5 mM PMSF) for 4 h at 4 °C and immobilized to protein G-Sepharose. Measurement of UV-induced Photoproducts in Genomic DNA by Slot-blot Analysis—Cells were irradiated with 12 J/m2 of UV-C light and incubated for various times. Antibodies bound to CPDs and []PPs were detected using the ECL plus Western blotting detection reagents (GE Healthcare) and analyzed by ImageQuant LAS-4000 (GE Healthcare)
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