Studying the DNA damage response in embryonic systems.
Lo Furno E, Recolin B, van der Laan S, Aze A, Maiorano D
Genome Surveillance and Stability
Molecular bases of human diseases
Our group is generally interested in the regulation of the DNA Damage Response (DDR), and in particular in its regulation during the early steps of embryonic development. The main function of the DDR is to slow down, or arrest cell prolferation in the presence of DNA lesions (DNA breaks, telomer integrity, replication forks stall) so to avoid cell division in the presence of DNA damage and therefore avoid the propagation of mutations that are drivers of genomic instability. A strong genomic instability is a feature of cancer cells (telomeric fusions, trabnsocations, duplications, deletions). To date it is admitted that the majority of sporadic tumours are the consequence of mutations in DDR genes, which can also lead to the development of genetic diseases. Hence the DDR is currently considered as a major barrier to malignat transformation playing a key role in maintenace of genomic stability.
PUBLICATIONS OF THE TEAM
Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos
Lo Furno E., Busseau, I., Aze, A., Lorenzi, C., Saghira, C., Danzi, M., Zuchner, S., Maiorano, D.
Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress.
Maiorano D, El Etri J, Franchet C, Hoffmann JS
Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites
Jihane Basbous, Antoine Aze, Laurent Chaloin, Rana Lebdy, Dana Hodroj, Cyril Ribeyre, Marion Larroque, Caitlin Shepard, Baek Kim, Alain Pruvost, Jérôme Moreaux, Domenico Maiorano, Marcel Mechali, Angelos Constantinou
Recent advances in understanding DNA replication: cell type-specific adaptation of the DNA replication program.
Aze A, Maiorano D
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy
Benkafadar N, Menardo J, Bourien J, Nouvian R, François F, Decaudin D, Maiorano D, Puel JL, Wang J.
Regulation of DNA Replication in Early Embryonic Cleavages
Kermi, C., Lo Furno, E., Maiorano, D
An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism
Hodroj D, Recolin B, Serhal K, Martinez S, Tsanov N, Abou Merhi R, Maiorano D
Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells
Hodroj, D., Serhal, K., Maiorano, D.
Chapter 6 -Open Access - Genomic instability of pluripotent stem cells: origins and consequences
Lo Furno, E., van der Laan, S., and Maiorano, D.
Regulation of translesion DNA synthesis by PCNA monoubiquitylation and beyond
Tsanov, N., Kermi, C., Delgado, J., Serrano, L., Maiorano D.
Rad18 is a maternal limiting factor that suppresses the UV-dependent DNA damge checkpoint in Xenopus embryos
Kermi, C., Prieto, S., van der Laan, S., Tsanov, N., Recolin, B., Uro-Coste, E., Delisle, M-B., and Maiorano, D.
Checking the cycle by ERRβ splice variants
Vanacker JM, Maiorano D.
PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase h and k focus formation upon UV damage
Tsanov, N., Kermi, C., Coulombe, P., Van der Laan, S., Hodroj, D., Maiorano, D.
Post-translational modifications in embryonic cell cycle
Van der Laan, S., Maiorano, D.
Cell Cycle-Dependent Expression of Dub3, Nanog and the p160 Family of Nuclear Receptor Coactivators (NCoAs) in Mouse Embryonic Stem Cells
van der Laan S, Golfetto E, Vanacker JM, Maiorano D.
Molecular mechanisms of DNA replication checkpoint activation
Recolin B, van der Laan S, Tsanov N, Maiorano D.
DNA polymerase k-dependent DNA synthesis at stalled replication forks is important for Chk1 activation
Bétous R., Pillaire, M-J, Pierini, L., Van der Laan, S., Recolin B., Ohl-Séguy, E., Guo, C., Niimi, N., Gruz, P., Nohmi, T. Friedberg, E., Cazaux, C., Maiorano, D* and Hoffmann J-S*. * corresponding authors
Polk in replication checkpoint
Maiorano, D., Hoffmann, JS.
High Dub3 expression in mouse ESC couples the G1/S checkpoint to pluripotency
Van der Laan, S., Crozet, C., Tsanov, N., and Maiorano, D
Role of RPA as sensor in activation of the S-phase checkpoint in Xenopus egg extracts
Recolin, B., Van der Laan, S., and Maiorano, D.
Implication of RPA32 phosphorylation in S-phase checkpoint signalling at replication forks stalled with aphidicolin in Xenopus egg extracts
Recolin, B., Maiorano, D
XRCC1 interacts with the p58 subunit of DNA Pola-primase and may coordinate DNA repair and replication during S phase.
Levy N, Oehlmann M, Delalande F, Nasheuer HP, Van Dorsselaer A, Schreiber V, De Murcia G, Ménissier-de Murcia J, Maiorano D, Bresson Anne.
Regulation of S phase during early embryonic Xenopus development
Recolin, B., Maiorano, D.
Geminin is cleaved by caspase-3 during apoptosis in Xenopus egg extracts
Auziol C, Mechali M, Maiorano D.
MCM proteins and DNA replication.
Maiorano D, Lutzmann M, Mechali M.
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Replication and Genome Dynamics
A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus.
Lutzmann M, Maiorano D, Mechali M.
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Replication and Genome Dynamics
Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts
Maiorano, D., Krasinska, L., Lutzmann, M. and Mechali M.
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Replication and Genome Dynamics
MCM8, a novel DNA helicase which is not required for licensing but functions during processive chromosomal replication in vertebratres.
Maiorano, D., Cuvier, O., Danis, E., and Mechali, M.
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Replication and Genome Dynamics
Identification of full genes and proteins of MCM9, a novel, vertebrate-specific member of the MCM2-8 protein family
Lutzmann, M., Maiorano, D., and Méchali, M.
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Replication and Genome Dynamics
A hypophosphorylated form of RPA34 is a specific component of pre-replication centers.
Françon, P. ; Lemaitre, JM., Dreyer, C. ; Maiorano, D. ; Cuvier, O. and Marcel Méchali.
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Replication and Genome Dynamics
Crystal Structure of the Coiled-coil Dimerization Motif of Geminin : Structural and Functional Insights on DNA Replication Regulation.
Thepaut, M., Maiorano, D., Guichou, JF., Auge, MT., Dumas, C., Méchali, M., and Padilla, A.
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Replication and Genome Dynamics
Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis.
Maiorano, D., Rul, W., and Marcel Mechali
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Replication and Genome Dynamics
Specification of a DNA replication origin by a transcription complex.
Danis, E., Brodolin, K., Menut, S., Maiorano, D., Girard-Reydet, C. and Marcel Méchali.
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Replication and Genome Dynamics
Repression of origin assembly in metaphase depends on inhibition of RLF-B/cdt1 by geminin.
Tada, S., Li, A., Maiorano, D., Méchali, M., and Blow, J.
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Replication and Genome Dynamics
Stepwise Regulated Chromatin Assembly of MCM2-7 Proteins.
Maiorano, D., Lemaître, J.M. and Méchali, M.
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Replication and Genome Dynamics
XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis
Maiorano, D., Moreau, J., and Méchali, M.
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Replication and Genome Dynamics
Initiation of DNA replication in eukaryotes : questioning the origin.
Françon, P., Maiorano, D. and Méchali, M.
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Replication and Genome Dynamics
Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus.
Coué, M., Amariglio, F., Maiorano, D., Bocquet, S. and Méchali, M.
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Replication and Genome Dynamics
Regulation of genome stability during early embryogenesis
It has been known for a longtime that the DDR is inefficient in the early embryos, however the reasons for this regulation and the underlying molecular bases are poorly understood. We have explored this issue in the early embryos of fruit fly Drosophila melanogaster, the clawed frog Xenopus laevis as well as in mouse embryonic stem cells (ES cells).
In Xenopus, we have discovered that the DDR is inefficient because the embryos efficiently by-pass DNA lesions by constitutive activation of the DNA damage tolerance pathway involving translesion DNA synthesis (TLS). This regulation limits replication fork stalling in front of DNA lesions and therefore DDR activation (Figure 1). We have explored the consequences of constitutive TLS activation on genome stability during embryonic development. We have observed a very high mutation rate as well as the presence of large DNA rearrangements that depend on TLS. We have also determined the consequences of such mutations throughout development in Drosophila. We have found that absence of the TLS DNA polymerase eta (Pol eta) during the earliest stages of embryonic development results in reduced viability at the larva stage, and in decreased genetic variability on pericentromeric heterochromatin in the adults. We also found that the mutagenic signature of TLS Pol eta identified in Drosophila is similar to that found in certain human cancers (Figure 2, Lo Furno et al., Nucleic Acids Research, in press). This constitutes a novel mechanism of genetic variation operating during very early development contributing to the individual genetic polymorphism and may explain the predisposition to develop cancer. These findings also pave the way to understand the molecular basis of genome instability observed in human embryos.
Figure 1. Constitutive activation of translesion DNA synthesis inhibts DDR activation during early Xenopus development. (Adapted from Kermi et al., Dev Cell 2015).
Figure 2. Upper panels, very early Drosophila and Xenopus embryos. Middle panel, embryonic mutational signature of TLS Pol eta. Lower panel, Heatmap showing the normalized relative contribution of TLS Pol eta mutational signatures from human tumors identified in COSMIC database.
Mouse embryonic stem cells (ES) also display an inefficient DDR for the G1/S checkpoint and by consequence show several signs of genomic instability. We have discovered that in these cells the G1/S checkpoint is inefficient because the critical G1/S regulator, the CDC25A protein phosphatase, is very abundant. We have also identifed the molecular bases of this abundance by showing that its stability depends upon the Dub3 ubiquitine hydrolase whose expression is under control of two pluripotency factors, Esrrb and Sox2 (van der Laan et al., 2013 Mol Cell and Figure 2). We are currently investigating the molecular basis of genomic instability of ES and iPS cells to improve their use in regenerative medicine.
Figure 3. Cartoon showing how Dub3 expression controls activation of the G1/S checkpoint and the pluripotent state of mouse ES cells.
Implication of translesion synthesis in the therapeutic resistance of cancer
We have shown that ectopic expression of Rad18 in human somatic cells is sufficient to constitutively activate translesion DNA synthesis and shut down the DDR, as observed in the early embryo (Figure 3). In these conditions cells shows acquired resistance to DNA damaging agents, including those currenlty used in the clinical, such as cisplatin. We have also observed a strong expression of Rad18 in cancer stem cells of the agressive brain tumor glioblastoma, a cancer that shows an extraordinary resistance to therapy. We are currently exploring the possiblity to use Rad18 as a novel target in the treatment of this cancer whose outcome is still very poor.
Figure 4. Ectopic Rad18 expression in somatic mammalian cells is sufficient to induce spontaneous translesion synthesis nuclear focus formation.
Identification of novel DDR genes
In the aim of identifying new DDR-responsive genes, we have developped an in vitro screen using protein extracts derived from Xenopus eggs and identified five genes candidates. One of these is the Ddx19 RNA helicase, previously implicated in the export of the mRNA from the nucleus into the cytoplasm. We have shown that Ddx19 translocates from the nuclear peryphery into the nucleus upon DNA damage (Figure 4). We have also unveiled a novel nuclear function for this enzyme in the resolution of aberrant RNA:DNA hybrid structures formed upon conflicts between replication and transcription, the so called R-loops (Hodroj et al., EMBO J 2017). We are now in the process of understanding the molecular basis of this novel function for the Ddx19 helicase.
Figure 5. Model showing the ATR-dependent Ddx19 function in nuclear R-loop resolution.
Subject: Molecular characterization of new factors involved in DNA damage tolerance
Hanane Mechri - Start October 2021 Supervisor: Domenico Maiorano Co-supervisor: Antoine Aze
Thesis proposal: Subject: Molecular characterization of new factors involved in DNA damage tolerance
Course and current status
Since April 2007. Group leader of the "Genome Surveillance and Stability" team at the Institute of Human Genetics of Montpellier (France). Biochemistry and Cell Biology of DNA damage and replication checkpoints.
2001. Staff researcher employed by INSERM at the CNRS Institute of Human Genetics of Montpellier (France).
1997-2001. Postdoctoral fellow at the Institute Jacques Monod (Paris, France), then at the Institute of Human Genetics of Montpellier (France). Biochemistry of DNA replication in Xenopus in vitro systems.
1996. Research Assistant at the University of Oxford.
1995. PhD at the University of Oxford (England, UK). Cell cycle regulation of DNA replication in fission yeast.
Membership
- Member of Trinity College, Oxford (England, UK)
- Member of Faculty of 1000 Biology “Nuclear Structure and Function Section”
- Member of the French Society of Cell Biology
- Biography published by Marquis “Who’s Who in the World”, “Who’s Who in Healthcare and Medicine”, “ Who’s Who in Science and Engineering”.
- Academic editor at PloS One
- Member of the editorial board of faculty of Faculty of 1000 Research
- Member of the French Society of Biochemistry and Molecular Biology (SFBBM)
