17 December

PhD defense Wouter Hoogenboom: Repair of DNA interstrand crosslinks

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Wouter Hoogenboom from group of Puck Knipscheer successfully defended his thesis “A high priority shipment – How nuclease delivery by SLX4 mediates DNA interstrand crosslink repair” on the 17th of December. During his PhD, Hoogenboom studied a dangerous form of DNA damage, the so-called interstrand crosslink, which arises when a reactive molecule connects the two strands of DNA. This type of DNA damage occurs naturally, but is also caused by chemotherapy because it helps kill tumor cells.

Schematic of interstrand crosslink repair

 

Every day our cells are confronted with thousands of DNA damage incidents in various forms. If this damage is not adequately repaired, this leads to mutations in the DNA that can cause genetic diseases. An exceptionally toxic form of DNA damage is the so-called “DNA interstrand crosslink” (ICL). In an ICL, the two DNA strands are connected to each other, so that they cannot separate. However, the separation of the two strands is necessary for important cellular processes, such as gene expression (the activation of genes) and DNA replication (doubling of all DNA before cell division). Unrepaired ICLs can result in cell death. This effect of ICLs is used in chemotherapy: ICL-inducing substances are used to kill the cancer cells.


Xenopus laevis
It has long been unclear why ICLs usually arise and how cells repair ICLs. To further study this process, Hoogenboom has used a model system based on protein extracts from eggs of the Xenopus laevis frog. This system uses pre-made ICLs, which means that the use of ICL-inducing substances, which also cause other types of DNA damage, is not necessary. This allowed him to specifically investigate the molecular mechanisms of the ICL repair that has been studied so sparsely until now.

Repair
The disconnection of the DNA strands is essential for repair. This is done by cutting one of the two strands and requires at least the protein SLX4 and the protein complex XPF-ERCC1. During his PhD, Hoogenboom investigated how SLX4 and XPF-ERCC1 ensure the disconnection of a DNA strand in the ICL repair process, at the molecular level. To investigate this, he made many different erroneous forms of SLX4 and XPF-ERCC1 in which each time a different piece of the proteins was mutated. This allowed him to investigate exactly which parts of these proteins are important for which parts of the repair process. XPF-ERCC1 is a protein complex that can cut the DNA, a step necessary for the repair of an ICL. By making different variants of this protein complex, he discovered that one specific part is responsible for the interaction with the protein SLX4 and that without this interaction repair cannot take place. He also discovered that specific pieces of the SLX4 protein are responsible for recruiting the XPF-ERCC1 protein complex to the right spot in the DNA, so that the DNA can be cut there. Finally, Hoogenboom also investigated how SLX4 is in turn recruited to the ICL and identified certain domains in SLX4 that appear to be responsible for this. However, exactly how these domains ensure that SLX4 is brought to the ICL in the DNA is not yet clear and needs further investigation in the future.

Resistance
Further investigation of the ICL repair mechanism can contribute to the development of substances that specifically inhibit ICL repair. This is useful because in some cases cancer cells build up resistance to chemotherapeutic agents by increasing the production of, for example, XPF-ERCC1. Such “inhibitors” could make cancer cells more sensitive to therapy while their side effects are limited.

 

 

Wouter Hoogenboom did his PhD research in the group of Puck Knipscheer, where he will continue to work as a postdoc after his defense.