Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

Abstract

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.

Histone Deacetylase Inhibitor Sodium Butyrate Enhances the Cell Killing Effect of Psoralen plus UVA by Attenuating Nucleotide Excision Repair

“…The mechanism by which ICL are repaired in mammals is not well understood; however, it seems to involve several DNA repair systems, such as NER, homologous recombination (HR) repair, translesion synthesis (TLS), and the Fanconi anemia/BRCA (FA/ BRCA) pathway (25,30,31). In our understanding based on a recently proposed mechanism for the repair of psoralen-ICL, NER is important for the early stages of the repair process: (a) recognition of ICL is probably carried out by NER proteins, such as XPE (also called DDB2 or p48), XPC-Rad23, and XPA-RPA (28,32,33) and (b) dual incision at both sides of the lesion is mediated by the NER excinuclease ERCC1/XPF, leading to an uncoupling of one arm of the ICL (34). Thereafter, TLS, FA/BRCA, and HR pathways would function sequentially.…”

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

“…Cisplatin forms a small number of ICL and fails to induce DSB in rodent cells [48]. NER processing of psoralen ICL that depends on XPA and other NER proteins to uncouple one strand of the ICL [21,22], followed by translesion synthesis with a bypass polymerase may be one scenario that could account for reduced DSB formation in GM637 cells relative to XP-A cells [11,20,50,51].…”

“…With the exception of cells lacking ERCC1 and XPF proteins, which are extremely sensitive to ICL-forming agents, other NER-deficient cells are only moderately sensitive, suggesting that the NER pathway is not critical to survive ICL formation [15,16]. On the other hand, cells deficient in the XPA protein, which is essential for NER, are defective in ICL removal [17][18][19][20], while XPA and another NER protein, XPC, appear to participate in the recognition of ICL associated with triplex-forming oligonucleotides [21,22]. Additionally, the XPF-ERCC1 heterodimer possesses functions independent of its role as a structure-specific 5′ endonuclease in NER [23].…”

Triple helix-interacting proteins and cancer

“…While the identities of several of these triplex-binding proteins are as yet unknown, for those that have been identified, many have known roles involving nucleic acids. Examples include the bacterial Tn7 protein and its role in transposon insertion 21 , yeast CDP1 and chromosome segregation 22 , Drosophila GAGA factor and transcriptional regulation 23 , human Orc4 protein and replication 24 , human XPA-RPA DNA repair complexes 25 , and murine HMG proteins and a variety of DNA-dependent processes (transcription, replication, recombination and repair) 26 . Note that identified triplex-binding proteins are not limited to those that interact with DNA.…”

“…Absence of these proteins has been found to increase genomic instability, especially at regions having the potential to form triplex structures 32 . Finally the nucleotide excision repair complex containing XPA can be directed to triplex structures by the associated protein RPA and thereby facilitate repair of proximal DNA photoadducts 25 . This repair, however, may only have tangential effects on triplex stability and is not the primary consequence of XPA-RPA function.…”