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The propagation of life relies on the accurate replication and distribution of genomes. When sister chromatids segregate in anaphase, pieces of DNA that connect them are stretched into structures that are known as anaphase DNA bridges. These bridges contain catenated, incompletely replicated, or damaged DNA and their resolution before cytokinesis is important to prevent the transmission of instable genomes to the arising daughter cells. We want to understand at a molecular and mechanistic level how an ensemble of DNA helicases, translocases, and topoisomerases recognizes and processes DNA bridges in dividing human cells.
Image: a HeLa cell getting ready to divide (courtesy of Stefano Maffini).
Biochemical reconstitution is a powerful tool to study the protein machinery that orchestrates a process as intricate as a mitotic cell division. We reconstitute isolated parts of the protein-DNA machinery from purified components and study their properties. The ability to then systematically mix and modify constituents and regulators enables meaningful biochemistry, structural biology, and single-molecule biophysics. We test the implications that emerge from our in vitro experiments in human cell lines.
Pim studied Molecular Life Sciences in Nijmegen, the Netherlands. He moved to Vienna in 2009 to study sister chromatid cohesion in the lab of Jan-Michael Peters at the IMP (PhD). In 2015, he joined the lab of Andrea Musacchio (MPI-MOPH, Germany) to investigate how kinetochores attach to the mitotic spindle. In August 2023, Pim started as a group leader at the Max Perutz Labs.
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We are happy with a small contribution to a very nice study by Colin Stok, Marcel van Vugt, and colleagues. The authors identified factors that become essential for viability in the absence of PICH (which processes anaphase DNA bridges). Among the 10 hits, there was 1 protein of unknown function that turns out to interact with FIGNL1 and RAD51 at replication forks. Alphafold predicted a very stable complex between FIGNL1 and FIRRM (check the pAE plot). Read the study here: [Stok et al., Cell Reports, 2023, https://doi.org/10.1016/j.celrep.2023.112668]
Ndc80 complexes are rod-like complexes that bind microtubules on one end and dock onto chromosomes at their other end. Using biochemistry, single-molecule biophysics, and cell biology, we discovered how a small sequence within the long coiled coil, known as the loop, promotes the formation of Ndc80 arrays on microtubules. This is important for chromosome congression during cell division, presumably because it allows for a cooperative attachment to the very end of a microtubule: [Polley et al., The EMBO Journal, 2023, https://doi.org/10.15252/embj.2022112504]
As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities and need to be recapitulated in vitro for the structural and functional characterization of mitotic protein machinery. In this paper, we describe how to get recombinant CDK1:Cyclin-B complexes that are efficiently phosphorylated at their T-loop, a prerequisite for kinase activity: [Huis in ’t Veld et al., Protein Science, 2022; https://doi.org/10.1002/pro.4233]
We engineered streptavidin scaffolds into an assembly with a single biotin binding site and up to nine covalently bound Ndc80 complexes. These spider-like objects span distances of well over a 100 nanometer. Collaborators at the TU Delft in the Netherlands attached these engineered Ndc80 multimers to the end of a homemade DNA origami nanospring to characterise their binding to dynamic microtubules: [Nick Maleki et al., Journal of Cell Science, 2022; https://doi.org/10.1242/jcs.260154]
Victor Cheng-chieh Chao
PhD Student +43 1 4277 74831
Room: 5.515
Pim Huis in 't Veld
Group Leader +43 1 4277 74830
Room: 5.515
Sara Landher
Master Student +43 1 4277 00000
Room: 5.515
Panagiotis Martzios
PhD Student +43 1 4277 74831
Room: 5.515
Saha Sayantan
PhD Student +43 1 4277 74831
Room: 5.515
Simona Smolkova
Master Student +43 1 4277 74831
Room: 5.515
FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics.
Stok Colin, Tsaridou Stavroula, van den Tempel Nathalie, Everts Marieke, Wierenga Elles, Bakker Femke J, Kok Yannick, Alves Inês Teles, Jae Lucas T, Raas Maximilian W D, Huis In 't Veld Pim J, de Boer H Rudolf, Bhattacharya Arkajyoti, Karanika Eleftheria, Warner Harry, Chen Mengting, van de Kooij Bert, Dessapt Julien, Ter Morsche Lars, Perepelkina Polina, Fradet-Turcotte Amelie, Guryev Victor, Tromer Eelco C, Chan Kok-Lung, Fehrmann Rudolf S N, van Vugt Marcel A T M
Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding.
Polley Soumitra, Müschenborn Helen, Terbeck Melina, De Antoni Anna, Vetter Ingrid R, Dogterom Marileen, Musacchio Andrea, Volkov Vladimir A, Huis In 't Veld Pim J
Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.
Huis In 't Veld Pim J, Wohlgemuth Sabine, Koerner Carolin, Müller Franziska, Janning Petra, Musacchio Andrea
Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling.
Huis In 't Veld Pim J, Volkov Vladimir A, Stender Isabelle D, Musacchio Andrea, Dogterom Marileen