Nucleotide Excision Repair (NER) in Eukaryotes

What is nucleotide excision repair?

Nucleotide excision repair (NER) is a highly conserved DNA repair pathway responsible for removing bulky DNA lesions that distort the helical structure of the double helix and stall DNA replication machinery. These lesions include cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, which are created by DNA bases absorbing ultraviolet (UV) light, as well as damage caused by environmental mutagens such as polycyclic aromatic hydrocarbons and certain chemotherapeutic agents. NER recognizes the structural abnormality of the lesions, rather than a particular mismatch in base sequence, enabling it to detect a broad range of chemically diverse DNA adducts.

NER is evolutionarily conserved, with mechanistically similar processes identified in bacteria (for example, the UvrABC system) and archaea, highlighting its essential role in genome maintenance. In eukaryotes, chromatin context plays an important role in repair efficiency; the compact nature of chromatin can hinder access to lesions, and specialized chromatin remodeling complexes, such as SWI/SNF, are recruited to open chromatin structure and facilitate NER factor binding.

Nucleotide excision repair functions throughout the cell cycle, excising and replacing damaged DNA segments, helping preserve genome stability and prevent accumulation of mutations that could otherwise disrupt replication or transcription. Through its critical role in maintaining DNA integrity, NER acts as a primary defense mechanism against mutagenesis and carcinogenesis in eukaryotic cells.  Deficiencies in NER are associated with disorders such as xeroderma pigmentosum and Cockayne syndrome.

Global genome NER (GG-NER) vs transcription-coupled NER (TC-NER)

Nucleotide excision repair is divided into two sub pathways. Global genome NER repairs DNA lesions throughout the genome, including non-transcribed regions, proactively scanning for DNA damage independent of transcription, and protecting genomic integrity. In contrast, transcription-coupled NER focuses on repairing DNA lesions in actively transcribed genes where RNA polymerase has stalled. Both pathways use a shared core repair machinery, but they differ in how the damage is recognized and the timing of their activity.

Steps in the nucleotide excision repair process

The nucleotide excision repair process is generally broken down into five steps: recognition of the damage, DNA unwinding, verification of the damage, dual incision of the damaged strand and finally resynthesis and ligation.

Damage recognition

The first step in nucleotide excision repair is recognizing the DNA lesion. In GG-NER, the XP-RAD23B complex scans the DNA throughout the genome, recognizing structural distortions. In TC-NER, when RNA polymerase II encounters a lesion during transcription, it stalls. Stalling triggers recruiting of Cockayne syndrome proteins CSA and CSB. For certain UV-induced lesions with less severe distortions, the UV-DDB complex (containing DDB1 and DDB2) facilitates lesion detection and handoff to XPC.

DNA unwinding

Once the damage has been recognized, the TFIIH complex is recruited. ATP-dependent helicases XPB and XPD unwind the DNA around the area of the lesion, creating an open bubble of approximately 25–30 nucleotides in eukaryotes.

Damage verification

Once the DNA is unwound, XPA is recruited to verify the damage, creating a scaffold for additional components needed to carry out the repair.

Dual incision of the damaged strand

Repair begins with coordinated nicking of the damaged strand on the 5’ side of the lesion by the XPF–ERCC1 complex and on the 3’ side by XPG. The excised fragment, typically 24–32 nucleotides in length, is removed, leaving a gap in the damaged strand.

Repair and ligation

In the final step of NER, DNA polymerase δ, ε or κ uses the template provided by the single strand to resynthesize the DNA, filling the gap with the correct sequence. Once the gap has been closed, DNA ligase seals the final nick in the DNA backbone, ensuring continuity of the strand.

Key proteins involved in the nucleotide excision repair pathway

Multiple proteins and complexes are involved in the nucleotide excision repair pathway. Key components include the XPC-RAD23B complex, Cockayne syndrome proteins CSA and CSB, transcription factor II H, RPA, XPA, the XPF-ERCC1 complex and XPG.

XPC-RAD23B complex

The XPC-RAD23B complex is the primary sensor of DNA damage in the GG-NER sub pathway of nucleotide excision repair. Xeroderma pigmentosum complementation group C (XPC) readily recognizes significant structural distortions of the DNA helix. Stabilized by RAD23B, XPC recruits TFIIH and downstream repair factors. When distortion of the DNA helix is less severe, XPC requires the help of the UV-DDB complex containing DDB1 and DDB2 to recognize the lesion.

CSA and CSB

Cockayne syndrome proteins CSA and CSB are recruited by stalled RNA polymerase at the site of DNA lesions encountered during transcription. They are the primary trigger of the TC-NER sub pathway of nucleotide excision repair, recruiting TFIIH and downstream repair proteins.

TFIIH

Transcription factor II H (TFIIH) is a multi-subunit complex containing XPB and XPD. Xeroderma pigmentosum type B (XPB) is a helicase that, once recruited to the site of damage, engages with DNA and opens the double helix. Xeroderma pigmentosum group D (XPD) is a 3’ to 5’ helicase that subsequently unwinds the DNA surrounding the lesion. Additional downstream components are subsequently recruited to carry out excision and repair.

RPA and XPA

Replication protein A (RPA) is a single-stranded DNA-binding protein that stabilizes the unwound DNA region generated by TFIIH helicase activity. It helps keep the DNA strand open, enabling accurate verification and excision of the lesion. RPA works with xeroderma pigmentosum group A protein (XPA), a DNA damage sensor that verifies presence of the DNA lesion and helps recruit the XPF–ERCC1 complex to the 5’ side of the lesion. RPA recruits XPG to the 3’ side of the lesion.

XPF–ERCC1 complex and XPG

Xeroderma pigmentosum group F (XPF) of the XPF-ERCC1 complex is an endonuclease that creates an incision on the 5’ side of the lesion, while Xeroderma pigmentosum group G (XPG) is an endonuclease that creates an incision on the 3’ side of the lesion. Working together, the damaged strand is removed, creating a single-stranded gap that is subsequently filled by DNA polymerase δ, ε, or κ, depending on the context, and sealed by DNA ligase I or III.

Health consequences of nucleotide excision repair dysfunction

Defects in NER lead to the accumulation of unrepaired DNA damage, genomic instability, and altered transcription, which can result in cancer, developmental abnormalities and premature aging.

Xeroderma pigmentosum

Mutations in genes that encode components of the nucleotide excision repair pathway cause Xeroderma pigmentosum, a rare genetic disorder characterized by extreme UV sensitivity and a highly increased risk of developing skin cancer. Increased cancer risk stems from the inability to repair UV-induced lesions, which leads to accumulation of mutations in oncogenes and tumor suppressor genes. Neurological symptoms are also associated with Xeroderma pigmentosum when NER is impaired in neurons.

Cockayne syndrome

Similar to Xeroderma pigmentosum, Cockayne syndrome is a rare genetic disorder that is caused by mutations in genes that encode components of the NER pathway. While transcription-coupled NER is compromised, global genome NER remains largely unaffected. As a result, patients don’t exhibit increased cancer risk. Symptoms are more focused on neurodevelopmental abnormalities, photosensitivity and features of premature aging with persistence of DNA lesions triggering apoptosis, cellular senescence and systematic inflammation.

Other NER-related syndromes

Trichothiodystrophy (TTD) results from mutations in certain TFIIH subunits and is characterized by brittle hair, developmental delays, and UV sensitivity. Unlike XP, TTD does not markedly increase cancer risk, illustrating how specific NER defects can yield distinct clinical outcomes.

References

1. Moreno NN, Olthof AM, Svejstrup JQ. Transcription-coupled nucleotide excision repair and the transcriptional response to UV-induced DNA damage. Annu Rev Biochem. 2023;92:81–113.
2. Piccione M, Belloni Fortina A, Ferri G, Andolina G, Beretta L, et al. Xeroderma Pigmentosum: General aspects and management. J Pers Med. 2021;11(11):1146.
3. Karikkineth AC, Scheibye-Knudsen M, Fivenson E, Croteau DL, Bohr VA. Cockayne syndrome: Clinical features, model systems and pathways. Ageing Res Rev. 2017;33:3–17.