Knipscheer: Genome Maintenance Back to research group The Knipscheer group studies molecular mechanisms of DNA replication and repair. Cellular processes that maintain the integrity of our genome are crucial to prevent genetic diseases such as cancer. The main interest of our laboratory is to decipher the molecular details of these processes. Currently, we focus on understanding how toxic DNA lesions, called interstrand crosslinks (ICLs), are repaired, and how stable secondary DNA structures (G-quadruplexes) are resolved. We study these processes in the context of active DNA replication and make use of Xenopus egg extracts that support highly regulated vertebrate DNA replication in vitro. In this experimental system, we can recapitulate both ICL repair and G-quadruplex replication in a test tube, allowing us to gain insights in their molecular mechanisms. DNA interstrand crosslink repair DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions that can form endogenously but are also induced at high doses in cancer chemotherapy. Repair of these lesions is complex and requires the collaboration of proteins from various DNA repair pathways including the Fanconi anemia (FA) pathway. FA is a genetic cancer-predisposition disorder caused by mutation in any of the 21 currently known FA genes. Cells from FA patients are sensitive to ICLs and we and others have shown that the FA pathway directly acts in ICL repair. We have recently shown that the FA pathway plays a pivotal role in a specific step in the repair process, the unhooking of the ICL from one of the DNA strands. These incisions require the activation of the FA pathway by ubiquitinylation of the FANCI-FANCD2 complex, the mediator protein FANCP/SLX4, and the endonuclease FANCQ/XPF-ERCC1 (Knipscheer et al., Science 2009, Klein Douwel, Boonen et al., Mol Cell 2014, Klein Douwel et al., EMBO J 2017). We are currently studying the molecular details of the incisions step and other aspects of ICL repair. G-quadruplex structure unwinding during DNA replication Our genome contains many G-rich sequences that can form stable secondary structures named G-quadruplex structures. Although these structures function in important biological processes (e.g. transcriptional regulation, telomere maintenance etc.), they have also been shown to induce DNA mutations. Our aim is to understand how G-quadruplexes are normally resolved to allow faithful DNA replication. Using a model system that combines Xenopus egg extracts and DNA templates containing specific G-quadruplex structures we have recently shown that replication stalls in close proximity of such a structure. This stalling is transient and G-quadruplexes are readily resolved and faithfully replicated in our system. Unwinding of a subset of G4 structures is strictly dependent on the FANCJ helicase (Castillo Bosch et al., EMBO J 2014). Currently, we are investigating which other factors are involved in this process and how this is regulated.
