Jeroen Essers

Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination.

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells. These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes.

Nuclear dynamics of PCNA in DNA replication and repair.

ABSTRACT The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks

Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

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.

Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination‐deficient mRAD54−/− mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double‐strand break repair through DNA end‐joining. In contrast, regardless of developmental stage, mRAD54−/− mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double‐strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation‐induced DNA damage changes during development of the animal.

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage.

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double‐strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi‐protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi‐protein complexes, rather than stable holo‐complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross‐talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

ABSTRACT We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.

Advanced Level-Set-Based Cell Tracking in Time-Lapse Fluorescence Microscopy

Cell segmentation and tracking in time-lapse fluorescence microscopy images is a task of fundamental importance in many biological studies on cell migration and proliferation. In recent years, level sets have been shown to provide a very appropriate framework for this purpose, as they are well suited to capture topological changes occurring during mitosis, and they easily extend to higher dimensional image data. This model evolution approach has also been extended to deal with many cells concurrently. Notwithstanding its high potential, the multiple-level-set method suffers from a number of shortcomings, which limit its applicability to a larger variety of cell biological imaging studies. In this paper, we propose several modifications and extensions to the coupled-active-surfaces algorithm, which considerably improve its robustness and applicability. Our algorithm was validated by comparing it to the original algorithm and two other cell segmentation algorithms. For the evaluation, four real fluorescence microscopy image datasets were used, involving different cell types and labelings that are representative of a large range of biological experiments. Improved tracking performance in terms of precision (up to 11%), recall (up to 8%), ability to correctly capture all cell division events, and computation time (up to nine times reduction) is achieved.

CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA‐PKcs (non‐homologous end‐joining). Genes of the non‐homologous end‐joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41–42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.