The DNA damage response (DDR) is a complex network of DNA repair processes and associated signaling mechanisms that maintains genome integrity by removing DNA lesions that are continuously induced by endogenous and exogenous sources. The intricate cooperation of all DNA repair and signaling mechanisms involved in the DDR determines how cells cope with DNA damage. In this thesis, particular attention is given to two major DNA repair pathways: the first is nucleotide excision repair (NER), which removes DNA-helix distorting lesions such as those induced by UV light. The second is interstrand crosslink repair (ICLR), which removes covalent linkages between bases on opposing DNA strands induced by DNA crosslinking chemicals and antitumor agents, such as cisplatin, psoralens and mitomycin C. The structure specific endonuclease ERCC1-XPF is essential to incise DNA next to lesions during both NER and ICLR. In humans, mutations in this complex give rise to hereditary DNA repair syndromes such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), xeroderma pigmentosum-Cockayne syndrome complex (XPCS), and Fanconi anemia (FA).
Most of the XPF patients carry heterozygous mutations, which makes it difficult to dissect the link between each XPF allele and its phenotypic consequences. Here, we investigate how XPF mutations reported in XP and XPCS patients functionally affect XPF activity in NER. By means of confocal imaging, genetic and proteomics approaches on human cells in culture we show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect repair capacity. In contrast, XPCS-causing mutation impair repair capacity, and are associated with persistent binding or continuous recruitment of XPF and the core NER machinery to UV-induced DNA damage. Moreover, our results suggest that different patient phenotypes associated with XPF mutations are dependent on the ability of the other XPF allele to function in NER.
While it is well known that engagement of ERCC1-XPF in NER is facilitated by binding to the key factor XPA, how its recruitment in ICLR is regulated is less clear. Therefore, we investigated how in human cells ERCC1-XPF is localized to different types of psoralen-induced DNA lesions, repaired by either NER or ICLR. We confirm its dependence on XPA in NER and furthermore show that its engagement in ICLR is dependent on FANCD2.
Hereditary DNA repair defects affect tissues differently, suggesting that in vivo cells can deal differently with DNA damage. Because defects found in ERCC-1/XPF patients, i.e. neurodegeneration, developmental defects and accelerated aging, are also observed in ERCC-1/XPF-1 deficient C. elegans, we used this model organism to study DDR in vivo. Our imaging approaches show that NER indeed displays differences depending on the cell type and suggest the existence of a tissue-specific organization of DDR. Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by a broad spectrum of clinical features. Interestingly, only half of the patients are non-photosensitive and carries mutations in TFIIEβ, involved in regulating transcription, RNF113, involved in activating the spliceosome, or MPLKIP/TTDN1, whose function is unknown. Using mass spectrometry analysis, DNA damage sensitivity assays and live cell imaging approaches, we show that TTDN1 function is likely linked to mRNA splicing and/or maturation as well as in DDR to ICLs and/or DNA double strand breaks.

Additional Metadata
Keywords DNA damage response, DNA repair, xeroderma pigmentosum Cockayne syndrome complex, Fanconi anemia, tissue-specificity, C. elegans
Promotor W. Vermeulen (Wim) , H. Lans (Hannes)
Publisher Erasmus University Rotterdam
ISBN 978-94-6332-535-6
Persistent URL hdl.handle.net/1765/120099
Citation
Sabatella, M. (2019, October 23). When a cut makes the difference : DNA damage incision from human cells to C. elegans. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/120099