Knipscheer: Genome Maintenance

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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.

Selected publicationsView all publications

Alcohol-derived DNA interstrand crosslinks are repaired by two distinct mechanisms.

Hodskinson MR*, Bolner A*, Sato K, […] Kind J, Chin J, Patel KJ#, Knipscheer P#

Nature. 579(7800):603-608

Download|2020

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair.

Sato K*, Brandsma I*, van Rossum-Fikkert SE, […] Knipscheer P#, Kanaar R#, Zelensky AN#

Nucleic Acid Research. 48(5);2442-2456

Download|2020

The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair.

Hoogenboom WS, Boonen RACM, Knipscheer P

Nucleic Acid Research. 18;47(5):2377-2388

Download|2020

Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair.

Klein Douwel D, Hoogenboom WS, Boonen RACM, Knipscheer P

EMBO J. 36(14):2034-2046

Download|2017

FANCJ promotes DNA synthesis through G-quadruplex structures.

Castillo Bosch P, Segura-Bayona S, […] Tijsterman M#, Knipscheer P#

EMBO J. 33(21):2521-33

Download|2014

XPF-ERCC1 acts in unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4.

Klein Douwel D*, Boonen RACM*, […] Walter JC, Knipscheer P

Mol Cell. 54(3):460-71

Download|2014

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis.

Brandsma I, Sato K, […] Knipscheer P#, Kanaar R#, Zelensky AN#

Cell Reports. 25;27(13):3790-3798

Download|2019

Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.

Hoogenboom WS, Klein Douwel D, Knipscheer P

Dev Biol. 428(2):300-309

Download|2017

Replication-coupled DNA interstrand cross-link repair in Xenopus egg extracts

Knipscheer P, Räschle M, Schärer OD, Walter JC

Methods Mol Biol. 920:221-43

Download|2012

The Fanconi anemia pathway promotes replication-dependent DNA interstrand crosslink repair.

Knipscheer P, Räschle M, […] Elledge SJ, Walter JC

Science. 326:1698-1701

Download|2009

Mechanism of replication-coupled DNA interstrand crosslink repair.

Räschle M, Knipscheer P, […] Schärer OD, Walter JC

Cell. 134:969-980

Download|2008

Group leader

Puck Knipscheer

Puck Knipscheer is group leader at the Hubrecht Institute, professor by special appointment of Biochemistry of Genome maintenance at the Leiden University Medical Center and Investigator at Oncode Institute. Her group studies how toxic DNA lesions, called DNA interstrand crosslinks (ICLs), are repaired and how secondary DNA structures (G-quadruplexes) are resolved. These processes are important to maintain the integrity of our genome which is crucial to prevent genetic diseases such as cancer. The Knipscheer group studies these processes during active DNA replication in Xenopus egg extracts. These extracts recapitulate both processes under physiological conditions, which enables them to study the molecular details of these important pathways.

Scientific training and positions