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Roland Kanaar

Publications and source records attributed to Roland Kanaar.

At least 19 recordsLinked to original sources
PubMed2025

A Reporter Platform to Study Therapy-Induced Senescence in Live Cancer Cells.

Senescence is a durable state of cell cycle arrest that can be induced by various stressors, including DNA damage caused by chemotherapeutic agents or ionizing radiation. In the context of cancer, therapy-induced senescence (TIS) plays a dual role: while it effectively halts tumor cell proliferation, TIS also carries the risk of promoting tumor relapse through the senescence-associated secretory phenotype (SASP). Beyond its direct impact on tumor cells, cancer therapies leading to TIS often induce short- and long-term side effects that significantly affect the quality of life for patients. However, the lack of universal biomarkers for TIS hinders a comprehensive understanding of its characteristics and its role in cancer therapies. A lamin-based senescence reporter platform is developed to reliably detect and sort live senescent cancer cells. This versatile tool supports live-cell imaging, enabling real-time tracking of senescence induction and escape to investigate heterogeneity in treatment response. Additionally, it allows high-content screening and marker integration, for example incorporating IL6 as SASP marker. It is therefore a valuable tool for fundamental research addressing new questions in the field of TIS as well as for drug discovery, including the development of novel senolytics.

Humans↗
PubMed2006

The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks.

Repair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81(-/-) Rad54(-/-) ES cells were as hypersensitive to ICL agents as Mus81(-/-) cells. We propose that Mus81-Eme1- and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.

Animals↗
PubMed2006

Architecture of a polycomb nucleoprotein complex.

Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (PHO) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of PHO and the multisubunit Polycomb core complex (PCC). Here, we analyzed the architecture of the PHO/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that PHO/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. We discuss the implications of these findings for models explaining PRE function.

Animals↗
PubMed2006

Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers.

Nucleoprotein filament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51 filament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein filaments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well-known (over)stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was approximately 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.

DNA↗
PubMed2006

DNA double-strand break repair and chromosome translocations.

Translocations are genetic aberrations that occur when a broken fragment of a chromosome is erroneously rejoined to another chromosome. The initial event in the creation of a translocation is the formation of a DNA double-strand break (DSB), which can be induced both under physiological situations, such as during the development of the immune system, or by exogenous DNA damaging agents. Two major repair pathways exist in cells that repair DSBs as they arise, namely homologous recombination, and non-homologous end-joining. In some situations these pathways can function inappropriately and rejoin ends incorrectly to produce genomic rearrangements, including translocations. Translocations have been implicated in cancer because of their ability to activate oncogenes. Due to selection at the level of the DNA, the cell, and the tissue certain forms of cancer are associated with specific translocations that can be used as a tool for diagnosis and prognosis of these cancers.

Chromosome Breakage↗
PubMed2006

Analysis of DNA double-strand break repair pathways in mice.

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.

Animals↗
PubMed2006

Multiple interlinked mechanisms to circumvent DNA replication roadblocks.

DNA replication is a fragile process, since unavoidable lesions in the template DNA cause replicative polymerases to stall, posing a serious threat to genome integrity. Homologous recombination, translesion DNA synthesis and de novo reinitiation of DNA synthesis ensure robust replication by navigating it passed damaged DNA. In this review, we highlight the relationship between these three processes.

Animals↗
PubMed2006

Analysis of DNA recombination and repair proteins in living cells by photobleaching microscopy.

DNA double strand break repair through homologous recombination has been shown biochemically to require the coordinated action of the RAD52 group of proteins, including the DNA strand exchange protein Rad51. We have started to develop experimental tools to investigate the close cooperation of homologous recombination proteins in living cells, where proteins operate in the context of chromatin and in the presence of other nuclear processes. This chapter describes in detail methods to establish cell lines stably expressing green fluorescent protein -tagged recombination proteins and photobleaching techniques to investigate the behavior of the proteins with the use of live cell video microscopy. Fluorescence recovery after photobleaching (FRAP), fluorescence loss after photobleaching (FLIP), and their combination in the same cell are useful techniques to gain insights into the dynamic behavior of the recombination proteins. Parameters such as diffusion rates and mobile versus immobile fractions before and after DNA damage induction can be obtained. In addition, residence times of recombination proteins at sites of DNA damage can be determined. Through the application of FRAP and FLIP it is possible to establish whether proteins are present in the same multiprotein complex, whether this is affected by DNA damage induction, and whether proteins dynamically associate with and dissociate from sites of DNA damage.

Animals↗
PubMed2006

Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis.

Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.

Animals↗
PubMed2006

DNA double-strand break repair: all's well that ends well.

Breaks in both DNA strands are a particularly dangerous threat to genome stability. At a DNA double-strand break (DSB), potentially lost sequence information cannot be recovered from the same DNA molecule. However, simple repair by joining two broken ends, though inherently error prone, is preferable to leaving ends broken and capable of causing genome rearrangements. To avoid DSB-induced genetic disinformation and disruption of vital processes, such as replication and transcription, cells possess robust mechanisms to repair DSBs. Because all breaks are not created equal, the particular repair mechanism used depends largely on what is possible and needed based on the structure of the broken DNA. We argue that although categorizing different DSB repair mechanisms along pathways and subpathways can be conceptually useful, in cells flexible and reversible interactions among DSB repair factors form a web from which a nonpredetermined path to repair for any number of different DNA breaks will emerge.

Animals↗
PubMed2005

Transcriptome analysis reveals cyclobutane pyrimidine dimers as a major source of UV-induced DNA breaks.

Photolyase transgenic mice have opened new avenues to improve our understanding of the cytotoxic effects of ultraviolet (UV) light on skin by providing a means to selectively remove either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts. Here, we have taken a genomics approach to delineate pathways through which CPDs might contribute to the harmful effects of UV exposure. We show that CPDs, rather than other DNA lesions or damaged macromolecules, comprise the principal mediator of the cellular transcriptional response to UV. The most prominent pathway induced by CPDs is that associated with DNA double-strand break (DSB) signalling and repair. Moreover, we show that CPDs provoke accumulation of gamma-H2AX, P53bp1 and Rad51 foci as well as an increase in the amount of DSBs, which coincides with accumulation of cells in S phase. Thus, conversion of unrepaired CPD lesions into DNA breaks during DNA replication may comprise one of the principal instigators of UV-mediated cytotoxicity.

Animals↗
PubMed2005

Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA.

The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.

Acid Anhydride Hydrolases↗
PubMed2005

Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function.

Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. We analyzed nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination.

Adenosine Triphosphate↗
PubMed2005

Torque-limited RecA polymerization on dsDNA.

The assembly of RecA onto a torsionally constrained double-stranded DNA molecule was followed in real time using magnetic tweezers. Formation of a RecA-DNA filament on the DNA tether was stalled owing to different physical processes depending on the applied stretching force. For forces up to 3.6 pN, the reaction stalled owing to the formation of positive plectonemes in the remaining DNA molecule. Release of these plectonemes by rotation of the magnets led to full coverage of the DNA molecule by RecA. At stretching forces larger than 3.6 pN, the twist induced during filament formation caused the reaction to stall before positive supercoils were generated. We deduce a maximum built-up torsion of 10.1 +/- 0.7 k(b)T. In vivo this built-up torsion may be used to favor regression of a stalled replication fork or to free the chromosomal DNA in E.coli from its condensing proteins.

Biopolymers↗
PubMed2005

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52.

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.

Animals↗
PubMed2005

Analysis of ionizing radiation-induced foci of DNA damage repair proteins.

Repair of DNA double-strand breaks by homologous recombination requires an extensive set of proteins. Among these proteins are Rad51 and Mre11, which are known to re-localize to sites of DNA damage into nuclear foci. Ionizing radiation-induced foci can be visualized by immuno-staining. Published data show a large variation in the number of foci-positive cells and number of foci per nucleus for specific DNA repair proteins. The experiments described here demonstrate that the time after induction of DNA damage influenced not only the number of foci-positive cells, but also the size of the individual foci. The dose of ionizing radiation influenced both the number of foci-positive cells and the number of foci per nucleus. Furthermore, ionizing radiation-induced foci formation depended on the cell cycle stage of the cells and the protein of interest that was investigated. Rad51 and Mre11 foci seemed to be mutually exclusive, though a small subset of cells did show co-localization of these proteins, which suggests a possible cooperation between the proteins at a specific moment during DNA repair.

Animals↗