Bernard S. Lopez

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

Plk3 phosphorylates CtIP in G1 in a damage-inducible manner and is required with CtIP for the repair of complex double-strand breaks and regulation of resection-mediated end-joining pathways.

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

Cellular uptake pathways of sepiolite nanofibers and DNA transfection improvement

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.

Genomic rearrangements induced by unscheduled DNA double strand breaks in somatic mammalian cells

DNA double‐strand breaks (DSBs) are highly toxic lesions that can lead to profound genome rearrangements and/or cell death. They routinely occur in genomes due to endogenous or exogenous stresses. Efficient repair systems, canonical non‐homologous end‐joining and homologous recombination exist in the cell and not only ensure the maintenance of genome integrity but also, via specific programmed DNA double‐strand breaks, permit its diversity and plasticity. However, these repair systems need to be tightly controlled because they can also generate genomic rearrangements. Thus, when DSB repair is not properly regulated, genome integrity is no longer guaranteed. In this review, we will focus on non‐programmed genome rearrangements generated by DSB repair, in somatic cells. We first discuss genome rearrangements induced by homologous recombination and end‐joining. We then discuss recently described rearrangement mechanisms, driven by microhomologies, that do not involve the joining of DNA ends but rather initiate DNA synthesis (microhomology‐mediated break‐induced replication, fork stalling and template switching and microhomology‐mediated template switching). Finally, we discuss chromothripsis, which is the shattering of a localized region of the genome followed by erratic rejoining.

The cohesin complex prevents the end-joining of distant DNA double-strand ends in S phase: Consequences on genome stability maintenance.

ABSTRACT DNA double-strand break (DSB) repair is essential for genome stability maintenance, but the joining of distant DNA double strand ends (DSEs) inevitably leads to genome rearrangements. Therefore, DSB repair should be tightly controlled to secure genome stability while allowing genetic variability. Tethering of the proximal ends of a 2-ended DSB limits their mobility, protecting thus against their joining with a distant DSE. However, replication stress generates DSBs with only one DSE, on which tethering is impossible. Consistently, we demonstrated that the joining of 2 DSBs only 3.2 kb apart is repressed in the S, but not the G1, phase, revealing an additional mechanism limiting DNA ends mobility in S phase. The cohesin complex, by maintaining the 2 sister chromatids linked, limits DSEs mobility and thus represses the joining of distant DSEs, while allowing that of adjacent DSEs. At the genome scale, the cohesin complex protects against deletions, inversions, translocations and chromosome fusion.

Sepiolite as a New Nanocarrier for DNA Transfer into Mammalian Cells: Proof of Concept, Issues and Perspectives

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier for DNA transfer thanks to its strong interaction with DNA molecules and its ability to be naturally internalized into mammalian cells through both non-endocytic and endocytic pathways. Sepiolite, due to its ability to bind various biomolecules, could be a good candidate for use as a nanocarrier for the simultaneous vectorization of diverse biological molecules. In this paper, we review our recent work, issued from a starting collaboration with Prof. Ruiz-Hitzky, that includes diverse aspects on the characterization and main features of sepiolite/DNA nanohybrids, and we present an outlook for the further development of sepiolite for DNA transfer.

The Cohesion complex maintains genome stability by preventing end joining of distant DNA ends in S phase

ABSTRACT Genome instability is a hallmark of cancer cells. The joining of distant DNA double-strand ends (DSEs) ineluctably leads to genome rearrangements. We found that the cohesion complex maintains genome stability by repressing the joining of distant DSEs specifically in the S phase, i.e., the main phase producing one-ended DSEs.

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein.

ABSTRACT Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations. Highlighted Article: The functional impact of missense mutations is difficult to predict. Yeast cells detect the misfolding effects of these types of mutations in the human BRCA1 protein, revealing an associated nuclear transport impairment.

Double-Strand Break Repair: Homologous Recombination in Mammalian Cells

Abstract Homologous recombination (HR) is an evolutionarily fundamental process that is essential for genome plasticity. HR participates in genome-stability maintenance while facilitating genetic diversity. This chapter summarizes the different molecular mechanisms of HR in mammals. The roles of HR in DNA double–strand break (DSB) repair, the reactivation of arrested replication forks, and their consequences (radiation sensitivity, meiosis, and application for genome manipulation) are discussed. HR competes with other pathways for DSB repair. Thus, a model is proposed for the choice of the DSB-repair pathway in two steps: (1) single-stranded DNA resection versus canonical nonhomologous end joining and (2) alternative end joining versus HR on resected DNA. HR is a double-edged sword that favors genome-stability maintenance but can generate genome instability. Situations of HR-induced genetic instability and strategies developed to protect against excess HR are presented: regulation during the cell cycle (HR is active in the S/G2 phase when the sister chromatids are present), the detoxification of abortive HR intermediates, and the repression of HR initiation. Then, HR misregulation in tumors and anticancer strategies targeting HR are discussed. Finally, this chapter presents the role of HR in the molecular evolution of multigene families in the process of concerted evolution.Homologous recombination (HR) is an evolutionarily fundamental process that is essential for genome plasticity. HR participates in genome-stability maintenance while facilitating genetic diversity. This chapter summarizes the different molecular mechanisms of HR in mammals. The roles of HR in DNA double–strand break (DSB) repair, the reactivation of arrested replication forks, and their consequences (radiation sensitivity, meiosis, and application for genome manipulation) are discussed. HR competes with other pathways for DSB repair. Thus, a model is proposed for the choice of the DSB-repair pathway in two steps: (1) single-stranded DNA resection versus canonical nonhomologous end joining and (2) alternative end joining versus HR on resected DNA. HR is a double-edged sword that favors genome-stability maintenance but can generate genome instability. Situations of HR-induced genetic instability and strategies developed to protect against excess HR are presented: regulation during the cell cycle (HR is active in the S/G2 phase when the sister chromatids are present), the detoxification of abortive HR intermediates, and the repression of HR initiation. Then, HR misregulation in tumors and anticancer strategies targeting HR are discussed. Finally, this chapter presents the role of HR in the molecular evolution of multigene families in the process of concerted evolution.