Research

Mechanistic function of RAD51 and its regulators

RAD51 protein functions as central mitotic recombinase forming helical nucleoprotein filament capable of homology search and strand invasion. Formation of RAD51 filament represents a key regulatory step during early phases of HR. RAD51 assembly and dissasembly is controlled by a variety of recombination modulators such as factors promoting filament formation and stimulating its activity, known as recombination mediators or proteins dismantling RAD51 presynaptic filament thereby preventing harmful recombination events, referred to as antirecombinases.

RAD51 filament is able to search for and invade homologous duplex DNA, and also protect the DNA from degradation at damaged replication forks, but relies on numerous co-factors (BRCA2, RAD51 paralogs, RAD51AP1) to drive the HR reaction to completion. However, a mechanistic understanding of how these co-factors act is unclear and remains a significant challenge to the field.

Through the integration of biochemistry and biophysics with structural biology and validation in biological systems, we aim to describe the mechanisms by which HR co-factors impact on the HR reaction and DNA replication. Deciphering these various regulations could hold the key to understanding the cancer-prone nature of mammalian cells, molecular mechanism of cancerogenesis and possibly unravel novel RAD51 filament-targeting strategies suitable for treatment of therapy-resistant tumours.

Selected publications:

Akita M, Girvan P, Spirek M, Novacek J, Rueda D, Prokop Z, Krejci L. Mechanism of BCDX2-mediated RAD51 nucleation on short ssDNA stretches and fork DNA. Nucleic Acids Res. 2024 Oct 28;52(19):11738-11752. doi: 10.1093/nar/gkae770

Špírek M, Taylor MRG, Belan O, Boulton SJ, Krejci L. Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function. Nat Commun. 2021 Sep 20;12(1):5545. doi: 10.1038/s41467-021-25830-x.

Taylor MRG, Špírek M, Jian Ma C, Carzaniga R, Takaki T, Collinson LM, Greene EC, Krejci L, Boulton SJ. A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling. Mol Cell. 2016 Dec 1;64(5):926-939. doi: 10.1016/j.molcel.2016.10.020

Taylor MRG, Špírek M, Chaurasiya KR, Ward JD, Carzaniga R, Yu X, Egelman EH, Collinson LM, Rueda D, Krejci L, Boulton SJ. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination. Cell. 2015 Jul 16;162(2):271-286. doi: 10.1016/j.cell.2015.06.015

Biomolecular condensates in genome maintenance

Biomolecular condensates are membrane-less cellular compartments that form by phase transitions of proteins and nucleic acids. These structures dynamically organize cell interior, confine and regulate biochemical processes, and serve as storage sites for proteins and RNAs. The regulated formation, maturation and dissolution of biomolecular condensates promotes genome integrity by spatial and temporal regulation of DNA repair pathways, DNA replication and transcription.

The molecular composition dictates chemical and physical characteristics of individual condensates, which in turn profoundly impact their function in cells. For instance, while condensates of certain proteins promote double-strand break repair by HR, a distinct type of condensates prevents undesirable recombination in highly repetitive regions of genome. Additionally, human pathologies (such as neurodegenerative diseases or leukemias) are often linked to aberrant formation of condensates with altered properties.

Our lab utilizes long experience with in vitro reconstitution of DNA repair pathways and translates it into the context of biomolecular condensates. We aim to use this bottom-up approach to reveal the molecular mechanisms by which the condensates regulate critical processes of genome maintenance.

Selected publications:

Merigliano C, Ryu T, Cibulka J, Rawal CC, See CD, Mitra A, Reynolds TW, Butova NL, Caridi CP, Li X, Wang J, Deng C, Chenoweth DM, Sung P, Capelson M, Krejčí L, Chiolo I. Off-pore Nup98 condensates mobilize heterochromatic breaks and exclude Rad51. Mol Cell. 2025 Jun 19;85(12):2355-2373.e11. doi: 10.1016/j.molcel.2025.05.012

Papageorgiou AC, Pospisilova M, Cibulka J, Ashraf R, Waudby CA, Kadeřávek P, Maroz V, Kubicek K, Prokop Z, Krejci L, Tripsianes K. Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase. Nat Commun. 2023 Oct 24;14(1):6751. doi: 10.1038/s41467-023-42503-z

RECQ helicases in DNA replication and repair

RECQ helicases are a conserved protein family involved in various aspects of genome maintenance, including most DNA repair pathways, replication and transcription. Humans have five different RECQ helicases that diverged substantially in both enzymatic activities (e.g. specificity for DNA substrates) and functions in cells. The importance of this family for genome integrity is underscored by pathogenic mutations in RECQ4, which cause rare autosomal recessive disorders, including Rothmund–Thomson syndrome (RTS), RAPADILINO syndrome, and Baller–Gerold syndrome, as well as by associations between RECQ4 overexpression and cancer.

Our lab currently focuses on RECQ4 protein, a unique member of its family with unusually broad spectrum of roles in genome maintenance. These range from involvement in distinct DNA repair pathways and initiation of DNA replication to functions at telomers or even in mitochondria. We use a combination of in vitro and in vivo approaches to decipher this complex functional landscape and relate it to the human pathologies associated with RECQ4 mutations.

Selected publications:

Ashraf R, Polasek-Sedlackova H, Marini V, Prochazkova J, Hasanova Z, Zacpalova M, Boudova M, Krejci L. RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome. Nat Commun. 2025 Feb 3;16(1):1302. doi: 10.1038/s41467-025-56518-1

Papageorgiou AC, Pospisilova M, Cibulka J, Ashraf R, Waudby CA, Kadeřávek P, Maroz V, Kubicek K, Prokop Z, Krejci L, Tripsianes K. Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase. Nat Commun. 2023 Oct 24;14(1):6751. doi: 10.1038/s41467-023-42503-z

Xue C, Molnarova L, Steinfeld JB, Zhao W, Ma C, Spirek M, Kaniecki K, Kwon Y, Beláň O, Krejci K, Boulton SJ, Sung P, Greene EC, Krejci L. Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates. Nucleic Acids Res. 2021 Jan 11;49(1):285-305. doi: 10.1093/nar/gkaa1184

Di Marco S, Hasanova Z, Kanagaraj R, Chappidi N, Altmannova V, Menon S, Sedlackova H, Langhoff J, Surendranath K, Hühn D, Bhowmick R, Marini V, Ferrari S, Hickson ID, Krejci L, Janscak P. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis. Mol Cell. 2017 Jun 1;66(5):658-671.e8. doi: 10.1016/j.molcel.2017.05.006

DNA repair nucleases and development of selective inhibitors

Nucleases play an essential role in various DNA repair pathways from processing the damaged nucleotide and extensive resection of the DNA ends to resolution of various DNA intermediates. For these reasons, nucleases comprise an integral part of many repair pathways and their inactivation results in the accumulation of DNA damage, consequentially leading to genomic instability and cancer.

On the other hand, overexpression of nucleases often results in cancer cells being resistant to chemo- or radiation therapy. Therefore, inhibiting nucleases will be beneficial to the sensitization of cancer cells to various anticancer therapies currently in use. In addition, since nucleases are downstream repair factors and present the possibility for inhibiting their enzymatic activities more specifically, their inhibitors are expected to be extremely useful in personalized medicine using the synthetic lethal approach.

Implementing a very interdisciplinary approach that integrates organic synthesis and medicinal chemistry with biochemistry, biophysics, molecular biology, and cell biology, we aim to develop potent and selective inhibitors of nucleases. These compounds will serve both as selective molecular probes to dissect nucleases function in genome maintenance and as potential leads for therapeutic development.

Selected publications:

Prochazkova J, Carbain B, Marini V, Nikulenkov F, Havel S, Akavaram N, Khirsariya P, Sisakova A, Cibulka J, Boudova M, Zacpalova M, Kalovska M, Rodrigues J, Daniel L, Brezovsky J, Azzalin C, Paruch K, Krejci L. Discovery of two structurally distinct classes of inhibitors targeting the nuclease MUS81 and enhancing efficacy of chemotherapy in cancer cells. JMchem, 2026. doi: 10.1101/2025.07.25.666773

Nikulenkov F, Carbain B, Biswas R, Havel S, Prochazkova J, Sisakova A, Zacpalova M, Chavdarova M, Marini V, Vsiansky V, Weisova V, Slavikova K, Biradar D, Khirsariya P, Vitek M, Sedlak D, Bartunek P, Daniel L, Brezovsky J, Damborsky J, Paruch K, Krejci L. Discovery of new inhibitors of nuclease MRE11. Eur J Med Chem. 2025 Mar 5;285:117226. doi: 10.1016/j.ejmech.2024.117226

Ashraf R, Polášek-Sedláčková H, Marini MV, Procházková J, Hašanová Z, Zacpalová M, Boudová M, Krejci L. RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome. Nat Commun, 2025, 16. doi: 10.1038/s41467-025-56518-1