Making the cut: How XPF-ERCC1 unhooks DNA interstrand crosslinks

Daisy Klein Douwel

Onderzoeksoutput: PhD Thesis (Proefschrift)PhD thesis (Proefschrift)

Samenvatting

DNA interstrand crosslinks (ICLs) are highly toxic lesions that bind both strands of the DNA helix together, which prevents the DNA from unwinding. This blocks important cellular processes such as DNA replication and transcription. ICL inducing agents were among the first chemotherapeutic drugs, making use of the toxic nature of ICLs especially for fast dividing cells. Today, ICL-inducing agents are still the main line of treatment for many cancers. The repair mechanisms have long been elusive, but in recent years we have begun to understand the pathways that underlie ICL repair. Much of this knowledge has come from studying Fanconi anemia (FA), a rare genetic disorder that is characterized by an extreme cellular sensitivity to ICL-inducing agents. Repair of an ICL is initiated during DNA replication when a replication fork stalls at the damaged lesion. Subsequently, the FA pathway cooperates with a variety of DNA repair proteins, such as nucleases, translesion polymerases and recombinases to coordinate ICL repair. A critical step in the repair process are the dual incisions on one of the DNA strands, to release (or ‘unhook’) the ICL, but the exact mechanism of unhooking and the nucleases directly involved in this step have remained elusive. To investigate this we have made use of the Xenopus laevis egg extract. This unique system can recapitulate the replication-dependent repair of a DNA plasmid with a single site-specific ICL. Using this cell free system we have discovered that the structure specific endonuclease XPF-ERCC1 is absolutely required for the unhooking incisions to take place. Furthermore, the recruitment of XPF-ERCC1 to the site of damage is dependent on interaction with SLX4/FANCP. Recruitment of both XPF-ERCC1 and SLX4 is in turn promoted by the ubiquitination of FANCD2, a key step in the activation of the FA pathway. We furthermore shed insight into the interaction of SLX4 and XPF, by identifying residues on XPF that are crucial for the interaction of the two proteins and therefor for ICL repair. Additionally, we investigated mutations found in XPF in patients with FA, and found that these residues were important for the repair of ICLs, while they did not affect the function of XPF-ERCC1 in nucleotide excision repair. This is another important DNA repair pathway that deals with UV damage and bulky lesions and requires XPF-ERCC1. These separation of function mutations have shed light on the differential regulation of XPF-ERCC1 in multiple repair pathways. Finally, we have set up a mass spectrometry based screen to identify novel factors required for ICL repair. Taken together, this research has greatly enhanced our understanding of the molecular mechanism of the repair of ICLs. A more extensive knowledge of the ICL repair pathway and the function of the proteins involved will enhance our understanding of Fanconi anemia, and could also lead to the identification of potential targets that can be used sensitize cells to chemotherapeutic agents.
Originele taal-2Engels
KwalificatieDoctor (dr.)
Toekennende instantie
  • University Utrecht
Begeleider(s)/adviseur
  • de Laat, Wouter, Promotor
  • Knipscheer, Puck , Co-promotor
Datum van toekenning19 jan 2017
Uitgever
Gedrukte ISBN's978-90-393-6703-2
StatusGepubliceerd - 19 jan 2017

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