Xeroderma Pigmentosum Complementation Group A Protein (XPA) Modulates RPA-DNA Interactions via Enhanced Complex Stability and Inhibition of Strand Separation Activity

Abstract

Replication protein A (RPA) participates in many cellular functions including DNA replication and nucleotide excision repair. A direct interaction between RPA and the xeroderma pigmentosum group A protein (XPA) facilitates the assembly of a preincision complex during the processing of DNA damage by the nucleotide excision repair pathway. We demonstrate here the formation of a ternary RPA, XPA, and duplex cisplatin-damaged DNA complex as is evident by electrophoretic supershift analysis. The RPA-XPA complex displays modest specificity for damaged versus undamaged duplex DNA, and the RPA-XPA complex displays a greater affinity for binding duplex cisplatin-damaged DNA when compared with the RPA or XPA proteins alone, consistent with previous results. Using DNA denaturation assays, we demonstrate that the role of XPA is in the stabilization of the duplex DNA structure via inhibition of the strand separation activity of RPA. Rapid kinetic analysis indicates that the bimolecular k on of the RPA-XPA complex is 2.5-fold faster than RPA alone for binding a duplex cisplatin-damaged DNA. The dissociation rate, k off , of the RPA-XPA complex is slower than that of the RPA protein alone, suggesting that the XPA protein stabilizes the initial binding of RPA to duplex DNA as well as maintaining the integrity of the duplex DNA. Interestingly, XPA has no effect on the k on of RPA for a single-stranded 40-mer DNA. Nucleotide excision repair (NER)1 is the major pathway responsible for the removal of a wide array of bulky DNA adducts from the genome (1, 2). A defect in this pathway can result in genomic instability and also results in a predisposition to skin cancer, as is evident by the xeroderma pigmentosum (XP) disorder (3). Although the protein components have been identified and the NER reaction has been reconstituted in vitro (4, 5), the biochemical process through which the global genomic repair pathway is initiated and recognizes damaged DNA is still poorly understood. The heterotrimeric replication protein A (RPA) is required for NER and has been suggested to play a role in the damage recognition process (6 -8). The XPA protein is also required for NER and is involved in the DNA damage recognition process (4, 5, 9). Both RPA and XPA preferentially bind damaged DNA, and because RPA and XPA directly interact in the absence of DNA, the RPA-XPA complex has been implicated as a key component in the earliest stage of damage recognition (9 -14). There is also evidence that the XPChHR23B protein complex may initiate recognition of DNA damage for the global genomic repair pathway of NER (15). Recent evidence also implicates the DDB heterodimer in damage recognition because the complex binds damaged DNA with high affinity (16) and can dramatically increase the repair rate of certain DNA adducts, including cyclobutane pyrimidine dimers in conjunction with XPA and RPA (17). In addition, the p48 subunit of DDB has been demonstrated to localize to the sites of UV-induced DNA damage independent of XPA and XPC (18).The exact role that the...

Xeroderma Pigmentosum Group A Protein Loads as a Separate Factor onto DNA Lesions

“…The same result was obtained with an XP-D/CS cell line bearing a mutation in the other helicase subunit, XPD of TFIIH (data not shown). These findings are in accordance with recent observations by Patrick and Turchi (47), who presented evidence that initial binding of RPA to the damaged DNA is subsequently further stabilized by an interaction with XPA. Whether XPA is incorporated in the NER complex in vivo in the absence of RPA remains to be determined.…”

Binding Discrimination of MutS to a Set of Lesions and Compound Lesions (Base Damage and Mismatch) Reveals Its Potential Role as a Cisplatin-damaged DNA Sensing Protein

“…Indeed, the competition of two proteins for a platinated DNA substrate has recently been correlated to their rates of association. Box B of the high mobility group box protein 1 (HMGB1) whose k on is close to the diffusion limit (Ϸ10 9 M Ϫ1 s Ϫ1 ) (56) selectively binds to a 1,2-d(GpG) intrastrand cross-link in the presence of human replication protein A (57), a DNA damage recognition protein in the nucleotide excision repair pathway that associates with this lesion at a rate that is about 2 orders of magnitude lower (58). In the present study our SPR assay demonstrated a k on of 3.1 ϫ 10 4 M Ϫ1 s Ϫ1 for MutS binding to the 1,2-d(GpG) intrastrand cross-link in agreement with values published for MutS and eukaryotic MutS␣ interacting with various DNA substrates (50,59,60).…”

Nucleotide excision repair deficient mouse models and neurological disease

“…The XPB and XPD subunits are 3′-5′ and 5′-3′ helicases, respectively, and unwind the DNA around the lesion. Subsequently, XPA and RPA are recruited to the site and help stabilize the open structure [6]. XPA also facilitates recruitment of ERCC1-XPF nuclease, which incises the damaged strand of DNA 5′ to the lesion, at the juncture between double-strand and single-strand DNA of the open complex.…”