The molecular mechanisms of nucleotide excision repair
The genetic information of our cells is stored on DNA in the cellular genome. Preservation and faithful transmission of this information is essential for cellular viability and human health. However, cellular DNA is continuously exposed to damaging agents and processes that can induce alterations ranging from base modifications to breaks in the DNA backbone. Therefore, DNA repair processes have evolved to repair damage inflicted on cellular DNA. We study one of these processes, termed Nucleotide Excision Repair (NER). NER responds to chemical modifications of DNA bases - including cross-linked bases and bulky adducts - caused by UV irradiation or reactive chemicals.
NER is a complex multi-step pathway in which DNA damage is sensed either by stalling of RNA polymerase II (transcription-coupled NER) or by specialised damage-sensing proteins (global genome NER). Upon damage detection, a large multi-protein complex called TFIIH is recruited, unwinds the DNA, and provides access to DNA endonucleases that excise the stretch of DNA that harbours the damaged nucleotides. Subsequently, DNA polymerases synthesise new DNA to close the resulting gap in one of the DNA strands.
NER is a complex multi-step pathway in which DNA damage is sensed either by stalling of RNA polymerase II (transcription-coupled NER) or by specialised damage-sensing proteins (global genome NER). Upon damage detection, a large multi-protein complex called TFIIH is recruited, unwinds the DNA, and provides access to DNA endonucleases that excise the stretch of DNA that harbours the damaged nucleotides. Subsequently, DNA polymerases synthesise new DNA to close the resulting gap in one of the DNA strands.
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(A) The cryo-EM structure of human TFIIH, comprising the core and CAK subcomplexes.
(B) Simplified schematic of global genome NER. Greber BJ, Toso DB, Fang J, Nogales E (2019). The complete structure of the human TFIIH core complex. eLife 8: e44771. PubMed Greber BJ, Perez-Bertoldi JM, Lim K, Iavarone AT, Nogales E (2020). The cryoelectron microscopy structure of the human CDK-activating kinase. Proc. Natl. Acad. Sci. U.S.A. 549 (7672): 414-417. PubMed |
Mutations in components of the NER pathway cause human diseases, including the DNA repair syndrome xeroderma pigmentosum as well as the combined transcription and DNA repair syndromes Cockayne syndrome and trichothiodystrophy. Furthermore, the action of NER can remove DNA lesions induced by chemotherapeutics, implicating the pathway in therapy resistance. Both the function and dysfunction of NER are thus of great importance to human health.
We aim to uncover the mechanisms of NER by applying structural, biochemical, and biophysical methods. In particular, we are interested in describing the structural transitions that occur within large multi-protein NER complexes, investigate how these transitions are regulated, and obtain insight into the mechanisms by which mutations impair the process and cause disease. One of the key techniques employed in our work is cryogenic electron microscopy (cryo-EM), which not only allows us to obtain detailed high-resolution information of the complexes under study but also enables analysis of the conformational landscapes of dynamic molecular assemblies. For more details on cryo-EM and the challenges this work entails, please have a look at our section on high-resolution cryo-EM.
We aim to uncover the mechanisms of NER by applying structural, biochemical, and biophysical methods. In particular, we are interested in describing the structural transitions that occur within large multi-protein NER complexes, investigate how these transitions are regulated, and obtain insight into the mechanisms by which mutations impair the process and cause disease. One of the key techniques employed in our work is cryogenic electron microscopy (cryo-EM), which not only allows us to obtain detailed high-resolution information of the complexes under study but also enables analysis of the conformational landscapes of dynamic molecular assemblies. For more details on cryo-EM and the challenges this work entails, please have a look at our section on high-resolution cryo-EM.
De-repression of TFIIH during initiation of NER
The TFIIH core complex contains two SF2-type ATPase enzymes: The DNA helicase XPD, which serves for DNA unwinding during NER, and the DNA translocase XPB, which aids in promoter opening in transcription and cooperates with XPD in DNA bubble opening in NER. Prior biochemical and structural analysis have shown that the activity of the DNA helicase XPD is inhibited in the free form of the complex. Presence of the MAT1 subunit of the CAK subcomplex at the XPD ARCH domain leads to closure of the DNA channel, and the protein p62 occupies crucial functional interfaces, including the ATP binding pocket of XPD. This likely serves to prevent unwanted DNA unwinding in context where this could be problematic and potentially harmful, such as in transcription initiation.
During NER, the activity of XPD is required, and the enzyme thus has to be de-repressed during the early stages of NER. We are interested in understanding the molecular mechanisms by which this occurs and how other NER factors play a role in mediating XPD de-repression during NER.
During NER, the activity of XPD is required, and the enzyme thus has to be de-repressed during the early stages of NER. We are interested in understanding the molecular mechanisms by which this occurs and how other NER factors play a role in mediating XPD de-repression during NER.
XPF-ERCC1 in SLX4-dependent DNA repair pathways
Many NER factors are multifunctional. TFIIH, with its functions in transcription, NER, and (via the CAK subcomplex) in CDK activation, is perhaps the best-known example. Similarly, the endonuclease XPF-ERCC1 not only serves to cut out damaged DNA segments in NER, but also functions in other DNA repair and genome maintenance pathways. These include interstrand crosslink repair, homologous recombination, and alternative lengthening of telomeres. In these roles, XPF-ERCC1 is recruited to molecular complexes that contain the giant SLX4 scaffold. SLX4 contains binding sites for several proteins or protein complexes involved in DNA repair and genome maintenance, including the three endonucleases XPF-ERCC1, MUS81-EME1, and SLX1.
To better understand how XPF-ERCC1 is targeted to SLX4-dependent DNA repair pathways and how this impacts its enzymatic activity, we determined cryo-EM structures of XPF-ERCC1 in complex with SLX4 and the DNA repair and genome maintenance factor SLX4IP. In combination with biochemical experiments, these structures show that a short segment of SLX4 is sufficient for complex formation with XPF-ERCC1, and that an SLX4 fragment comprising residues 330-555 can stimulate the enzymatic activity of XPF-ERCC1. Furthermore, while it was previously unclear whether SLX4IP binds to SLX4, XPF-ERCC1, or both, our structures demonstrate that bind to XPF-ERCC1 independently.
To better understand how XPF-ERCC1 is targeted to SLX4-dependent DNA repair pathways and how this impacts its enzymatic activity, we determined cryo-EM structures of XPF-ERCC1 in complex with SLX4 and the DNA repair and genome maintenance factor SLX4IP. In combination with biochemical experiments, these structures show that a short segment of SLX4 is sufficient for complex formation with XPF-ERCC1, and that an SLX4 fragment comprising residues 330-555 can stimulate the enzymatic activity of XPF-ERCC1. Furthermore, while it was previously unclear whether SLX4IP binds to SLX4, XPF-ERCC1, or both, our structures demonstrate that bind to XPF-ERCC1 independently.
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(A) Cryo-EM map of the XPF-ERCC1-SLX4-SLX4IP complex. SLX4 and SLX4IP are visualised on opposite sides of XPF-ERCC1 and do not directly interact with each other.
(B) Details of the interaction between XPF-ERCC1 and SLX4. This minimal XPF-binding fragment of SLX4 comprises residues 526-552. (C) SLX4 bound to XPF-ERCC1. The structure of SLX4IP was previously unknown. Our work shows that SLX4IP binding to XPF leads to conformational changes in XPF. Feng J, Martin PR, Kowalski S, Lecot M, Cronin NB, Matthews-Palmer T, Niedzwiedz W, Greber BJ# (2026) Molecular basis of XPF-ERCC1 targeting to SLX4-dependent DNA repair pathways. Nat. Commun. 17: 522. Link |
The addition of a DNA substrate to this complex led to the visualisation of bound DNA and additional segments of SLX4 on the surface of XPF-ERCC1, suggesting that these structural elements of SLX4 might be connected to the enzymatic activation of XPF-ERCC1. Indeed, biochemical experiments demonstrated that truncation of these segments of SLX4 led to diminished activation of XPF-ERCC1. Our results thus indicate that SLX4 contains a minimal interacting module that is required for XPF-ERCC1 binding as well as separate structural elements that enable full activation of XPF-ERCC1.
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(A) Cryo-EM map of DNA-bound (left) and apo (right) XPF-ERCC1-SLX4-SLX4IP complexes reconstructed from the same cryo-EM grid. Additional segments of SLX4 are visualised in the DNA-bound state.
(B) Schematic of the structural elements of SLX4 tested for their ability to enhance XPF-ERCC1 activity in our biochemical assays. (C) Endonuclease assay of XPF-ERCC1-SLX4IP bound to different SLX4 constructs. Removal of SLX4 residues 378-499 leads to diminished SLX4-dependent stimulation of XPF-ERCC1 activity. |
In summary, our data show how XPF-ERCC1 is targeted to SLX4-dependent DNA repair pathways, how SLX4 stimulates the endonuclease activity of XPF-ERCC1, and how SLX4IP interacts with XPF-ERCC1 to promote genome stability.
