The role of SLX4 in DNA interstrand crosslink repair

Researchers from the Knipscheer group have further elucidated the role of SLX4 in the repair of DNA interstrand crosslinks. If unrepaired, these crosslinks can lead to cell death. Mutations in SLX4 make cells more susceptible to DNA interstrand crosslinks and can cause Fanconi anemia, a genetic disease that often leads to bone marrow failure and predisposes the patient to cancer. The researchers found that specific regions of SLX4 are essential for its function in DNA interstrand crosslink repair, while other regions are dispensable for this function. Their results were published in Nucleic Acids Research on the 21^st of December.

Every day our cells are faced with the tremendous task of dealing with many thousands of incidences of DNA damage that appear in different forms. If these are not repaired properly this results in DNA mutations which can lead to genetic diseases. The DNA interstrand crosslink (ICL) is a particularly toxic type of DNA damage that sticks the two strands of the DNA together, making it impossible for the strands to separate. Separation of the DNA strands is necessary for important cellular processes, including gene expression and DNA replication. Unrepaired ICLs often lead to cell death, a trait that is exploited in cancer chemotherapy by using ICL inducing agents to kill cancer cells.

In higher organisms a mechanism has evolved that is specialized in repairing ICLs. This mechanism is initiated during DNA replication, when progression of the replication machinery is blocked by the crosslink. This is followed by a complex repair process that involves the Fanconi anemia pathway that currently counts 22 protein players. In comparison with many other DNA repair pathways, this ICL repair pathway is remarkably poorly understood.

SLX4 recruits the endonuclease XPF-ERCC1 to ICLs to make a cut in the DNA and unhook one strand from the ICL, which is required for ICL repair. Other SLX4-associated endonucleases are dispensable for this process, but an unknown factor is possibly responsible for a second cut.

A critical step in this repair process is the ‘unhooking’ of the crosslink from one of the DNA strands by cutting the DNA on either side of the lesion. This process requires endonucleases, proteins that can cut DNA. Recently, several details about the function of an essential endonuclease in this process, XPF-ERCC1, were uncovered by the Knipscheer group and others. However, the role of SLX4, a large docking protein that recruits three endonucleases, including XPF-ERCC1, to various DNA damage sites, remained largely unknown.

Using a model system based on protein extracts made from the eggs of the Xenopus laevis frog, researchers from the Knipscheer group studied the biochemical role of SLX4 in ICL repair. This system uses premade, chemically defined, and sequence specific DNA interstrand crosslinks and avoids the use of ICL inducing agents that induce a variety of DNA damage. This allows the researchers to exclusively investigate the molecular mechanism of this understudied DNA repair pathway.

They found that removal of SLX4 from the model system prevented ICL repair, while addition of purified SLX4 protein restored ICL repair. Similarly, several purified mutated SLX4 proteins were added to evaluate their ability to restore ICL repair. Using this approach they discovered that two-thirds of the full-length SLX4 protein could be removed without losing the ICL repair capacity. The dispensable region contains many sites for interactions with other proteins, including two endonucleases. This drastically confines the function of SLX4 in the process of ICL repair. In addition, the researchers found that the interaction of SLX4 with the endonuclease XPF-ERCC1 is crucial for crosslink repair, emphasizing the importance of recruiting this endonuclease to the proper site.

This study provides novel insights into the mechanism by which this toxic DNA damage is resolved. A better understanding of this mechanism could aid the development of novel therapeutic strategies in cancer treatment. ICL repair factors can serve as biomarkers for treatment with ICL inducing agents. In addition, specific ICL repair inhibitors could be developed to make treatment more efficient, to counteract resistance to treatment, or to exploit treatment options based on synthetic lethality.

Wouter S. Hoogenboom, Rick A.C.M. Boonen and Puck Knipscheer. The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair. Nucleic Acids Research 2018.

Puck Knipscheer is group leader at the Hubrecht Institute and Oncode Investigator.