Nucleotide excision repair (NER) is a vital cellular mechanism that repairs damaged DNA. It plays an essential role in maintaining genomic integrity, protecting the cell from various external and internal factors that can damage DNA, such as ultraviolet radiation, chemicals, and environmental toxins. The NER pathway is a multi-step process that involves a large number of proteins. The first step involves the recognition and binding of the DNA lesion by a complex of proteins called the XPC complex. This complex recognizes various types of DNA damage, including those caused by UV radiation and chemical agents. Once the damage is identified, the XPC complex recruits additional proteins, including the transcription factor TFIIH, which unwinds the DNA around the damage site.
The next step in the NER pathway involves the actual excision of the damaged DNA. This is carried out by a complex of proteins, including the endonucleases XPG and ERCC1-XPF, which make incisions on either side of the damage site, releasing a short oligonucleotide containing the damaged DNA. DNA polymerase and ligase then fill in the resulting gap, restoring the DNA to its original sequence.
NER is an essential repair pathway, as it is capable of correcting many types of DNA damage, including bulky adducts, pyrimidine dimers, and inter-strand cross-links. Defects in NER have been linked to several human genetic disorders, including xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. These disorders are characterized by hypersensitivity to UV radiation and other DNA-damaging agents and are associated with a significantly increased risk of cancer. In addition to genetic disorders, NER has also been implicated in the development of certain types of cancer. For example, mutations in the genes that encode the NER proteins XPC and XPD have been linked to an increased risk of skin cancer, while mutations in the ERCC2 gene have been associated with an increased risk of breast and lung cancer. The importance of NER is further underscored by the fact that it is conserved across all domains of life, including bacteria, archaea, and eukaryotes. This suggests that the repair pathway is fundamental to the survival of all living organisms.
While NER thus counteracts cancer formation, it is also important for antitumor therapy. Many antitumor agents, such as cisplatin, induce damage to DNA that can be repaired by NER. In this context, NER is a drug target of interest to improve cancer therapy outcomes. At the molecular level, NER is a highly complex and dynamic molecular machine, involving over 30 proteins that assemble at DNA lesions to excise the damage and replace it with intact DNA. This process is guided by protein-protein and protein-DNA interactions.
In a new research paper published in Proceedings of the National Academy of Sciences, a team of researchers led by Professor Orlando Schärer explored these interactions. The team found that two key proteins in NER, namely xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins, are required for organizing the pre-incision complex in NER. They reported how the two contacts between XPA and RPA contribute to the NER reaction using a structural approach and mutations in XPA that disrupt the binding interface with both RPA32C and RPA70AB. Biochemical and cellular assays show synergistic contributions to the physical interaction between RPA and XPA and to overall NER activity. Using integrated structural modeling, they show that the two contacts between XPA and RPA can be engaged simultaneously and generate models of how they shape the NER preincision complex.
The two proteins XPA and RPA are responsible for the organization of the NER complex after it has found the damage in DNA. The new study compared mutant variants of these two proteins to investigate how the two proteins engage in a pivotal interaction for the NER pathway. Specifically the authors discovered that two interaction interfaces between XPA and RPA are critical for NER and have distinct roles in the pathway. The interaction of XPA with RPA32 is crucial for the initial association of XPA with DNA damage, whereas the interaction between XPA and RPA70 is important for the completion of NER.
The research team conducted integrative structural studies of an XPA-RPA-DNA complex and revealed how the interactions of the two proteins shape the NER complex and trigger excision of the damage. The interaction of XPA and RPA32 occurs at the periphery of the complex, where it facilitates the initial assembly of the proteins at the site of damage. The interaction between XPA and RPA70 is located at the heart of the NER complex and forces the DNA into a U-shape. This allows the two ss/dsDNA junctions to become localized in close proximity, allowing for the NER complex to incise the DNA to remove the damage.
Understanding the importance of NER and its molecular mechanisms is critical to the development of new therapies for the treatment of genetic diseases and the prevention and treatment of cancer. In conclusion the new study revealed a surprising new model of the NER complex and how the interaction between XPA and RPA shapes its architecture. Disruption of the interaction between XPA and RPA inhibits NER, and the authors’ finding provides a blueprint for how this interaction may be targeted by small molecules to improve cancer therapy.
Mihyun Kim et al, Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair, Proceedings of the National Academy of Sciences (2022). DOI: 10.1073/pnas.2207408119