Nucleotide Excision Repair by Mutant Xeroderma Pigmentosum Group A (XPA) Proteins with Deficiency in Interaction with RPA

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 carcinogendamaged 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 Nterminal 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.

Structure of RPA32 bound to the N‐terminus of SMARCAL1 redefines the binding interface between RPA32 and its interacting proteins

“…A recent study has shown that a truncated XPA form with an N-terminal deletion of 29 amino acid residues reduces the binding to RPA32 protein but retains the interaction to RPA heterotrimer. However, compared with the wild-type protein, this truncated XPA form exhibits much lower DNA incision activity in cell-free NER assay, suggesting that the interaction of XPA and RPA32 is critical for NER repair [31]. Another interesting study has demonstrated that XPA can be acetylated at residues Lys63 and Lys67 [32], and deacetylation of XPA by SIRT1 promotes the interaction of XPA and RPA32.…”

Role of RPA Proteins in Radiation Repair and Recovery

“…Additionally, essential interactions between RPA and NER proteins are unaffected by hyperphosphorylation of RPA (see above) (Oakley, et al, 2003;Patrick, et al, 2005). Many of these repair proteins are capable of interacting with the unphosphorylated C-terminus of RPA2 rather than the Nterminus of RPA1, which may explain the separation of functions between unphosphorylated and hyperphosphorylated RPA (Ali, et al, 2010;Saijo, et al, 2011). Regardless of the mechanism, this demonstrates the versatility of RPA in DNA metabolism.…”

“…Nucleotide excision repair (NER) processes remove helixdistorting lesions by excising a patch of DNA, followed by re-synthesis. In this case, RPA physically interacts with the NER mediator XPA to direct incision to the damaged strand, and to help prevent excessive incision events, which could lead to further degradation of the genome (Krasikova, et al, 2010;Overmeer, et al, 2011;Saijo, et al, 2011). In mismatch repair (MMR), RPA and SSB interact with the exonuclease EXO1 to stimulate excision processes, as well as ensure correct excision termination (Genschel & Modrich, 2009;Lu & Keck, 2008).…”

The RASSF1A Tumor Suppressor Regulates XPA-Mediated DNA Repair

“…The complex is dynamic, and XPA enters the complex to help scaffold the cycle of repair proteins that enter and leave during the DNA repair process (15,37,38). One of the components of the complex that helps anchor XPA to DNA is the RPA trimer (39,40). As the RASSF1A SNP variant appeared to promote the retention of XPA in the nucleus after UV treatment, we wondered whether the SNPexpressing cells might demonstrate changes in the association of XPA with RPA.…”