Alumni: Dr. Arnab Ray Chaudhuri, Dr. Cindy Follonier Master Students: Isabella Zanini, Judith Oehler
Our research focuses on the molecular characterization of DNA replication stress and its contribution to genome instability. We aim to understand the mechanistic basis of genome rearrangements arising during perturbed DNA replication, affecting various aspects of human disease, such as cancer, aging and a growing number of neurodegenerative human syndromes. These studies take advantage of an established technological platform, ranging from standard molecular and cell biology methods to specialized single-molecule in vivo analysis of replication intermediates (DNA fibers/combing, psoralen-crosslinking coupled to electron microscopy).
Structural insights into oncogene-induced DNA replication stress
The DNA damage response is a critical anti-tumour barrier that prevents the proliferation of cells with potentially hazardous genetic alterations. It acts early in tumorigenesis and its activation was observed already in pre-cancerous lesions of various organs. The activation of the DNA damage checkpoint in these lesions was ascribed to oncogene-induced deregulation of DNA synthesis, or “replication stress”. Although the indirect consequences of replication stress, i.e. cell cycle arrest and senescence, have been elucidated to some extent, our understanding of the underlying molecular events is extremely vague. This is mainly due to the lack of information on the in vivo DNA structures generated under such conditions.
The replication stress phenotype can be reproduced in cell culture by overexpression of various oncogenes influencing DNA replication, e.g. Cyclin E, Cdc25A. We have exploited these systems to identify oncogene-associated defects in DNA replication. Overexpression of both oncogenes has a substantial effect on bulk DNA synthesis and leads to a marked slow-down of individual replication forks, measured by FACS analysis and DNA fiber labelling, respectively. Furthermore, electron microscopic analysis reveals the accumulation of aberrant replication intermediates upon induction of Cdc25A and CycE. However, only the overexpression of Cdc25A, and not of CycE, causes massive DNA breakage and full DDR activation shortly after oncogene induction (Figure 1). We found that Cdc25A-dependent DNA double strand breaks (DSB) are suppressed by preventing mitotic entry. We therefore propose that oncogene-induced replication stress promotes the accumulation of unusual replication intermediates and that oncogene-dependent DSB are due to premature activation of mitotic factors. We are currently testing the contribution in these phenomena of cellular factors that have been implicated in mitosis and/or are suspected to process replication intermediates (chromosome condensation, Holliday junction resolution). Moreover, we are exploiting a system recently established in the lab to investigate re-replication, a pathological phenomenon frequently associated with oncogene activation. Finally, we will extend our studies to more oncogenes and to their effect on primary cells.
Figure 1. Flow cytometric analysis of DNA synthesis, cell cycle progression and DDR activation after oncogene expression. (A) FACS-based distinction of γH2AX patterns after Cdc25A induction. Red and green signals indicate cells with pan-nuclear γH2AX and γH2AX foci, respectively. (B) FACS analysis after Cdc25A induction shows accumulation of cells with γH2AX foci and pan-nuclear staining. Pan-nuclear γH2AX is associated with replicative arrest. (C) FACS analysis after CycE induction shows early S-phase accumulation, followed by accumulation of cells in G2/M and checkpoint activation. At late timepoints, re-replicating cells with ≥4n DNA are detectable.
K. J. Neelsen, I. M. Y. Zanini, R. Herrador and M. Lopes (2013). Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates. Journal of Cell Biology, 200(6):699-708.
K. J. Neelsen, A. Ray Chaudhuri, C. Follonier, R. Herrador and M. Lopes (2013). Visualization and interpretation of eukaryotic DNA replication intermediates by electron microscopy in vivo. In "Functional Analysis of DNA and Chromatin", Humana Press, ed. J. C. Stockert. Methods in Molecular Biology, in press
Uncovering the structural determinants of DNA replication stress induced by cancer chemotherapeutics
DNA replication interference is one of the most common strategies employed in the clinics to kill actively proliferating cancer cells. TopoisomeraseI (Top1) can be trapped by specific inhibitors, such as Camptothecin or its clinically relevant derivatives Topotecan and Irinotecan, leading to interference with DNA metabolism and resulting in potent cytotoxicity in proliferating and cancer cells. Although replication-induced DSB have been consistently proposed to mediate this cytotoxicity, several recent reports challenge this view and propose a more complex coordination of replication fork progression in face of the topological stress induced by Top1-inhibition. Our single-molecule, biochemical and genomic studies in S. cerevisiae, mammalian cells and Xenopus egg extracts show that Top1 poisons rapidly induce replication fork slowing and reversal (Figure 2), which can be uncoupled from DSB formation at sublethal doses. Poly (ADP-ribose) polymerase activity, but not single strand break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a cellular strategy to prevent chromosome breakage upon exogenous replication stress and provide novel means to identify cellular factors that limit or mediate the cytotoxicity of anticancer drugs inducing replication stress. This important set of data has recently been published in Nature Structural and Molecular Biology. We are now committed to test the contribution of specific cellular factors, likely to play a role in formation, remodelling and/or resolution of reversed forks. We are particularly interested in testing in vivo the role of nuclease and helicase activities previously suggested to form or revert regressed forks. Among these we aim to identify PARP target proteins, as this could potentially explain the role of PARP in replication fork remodelling in face of stress. We are also assessing how the fine-tuning of Poly-ADP-ribosylation (via PARP and its antagonist protein PARG) contributes to fork structure and resistance to genotoxic stress. Furthermore, it will be particularly important to assess whether fork reversal is a specific response to Top1 poisoning or whether it entails a more general DNA transaction upon treatment with a wide range of cancer chemotherapeutics.
A. Ray Chaudhuri, Y. Hashimoto, R. Herrador, K.J. Neelsen,D. Fachinetti, R. Bermejo, A. Cocito, V. Costanzo and M. Lopes (2012). Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nature Structural and Molecular Biology19: 417–423
Berti*, A. Ray Chaudhuri*, S.
Thangavel, S. Gomathinayagam, S. Kenig, M.
Vujanovic, F. Odreman, T. Glatter, R. Mendoza-Maldonado, S. Graziano, B.
Lucic, V. Biasin, M. Gstaiger, R. Aebersold, J. M. Sidorova, R. J. Monnat, M.
Lopes° and A. Vindigni° (2013). Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition. Nature Structural and Molecular Biology, 20(3):347-354.
*equal contribution °corresponding authors
Structural analysis of DNA replication across unstable repetitive sequences
A growing number of human neurological hereditary diseases - among which Huntington disease, Freidreich's Ataxia and fragile-X are the most prominent - have been associated with trinucleotide repeat (TNR) expansion at various genomic loci. A large body of evidence suggests that these events are associated with DNA replication interference. Extensive studies in bacteria and yeast clarified that TNR can pause DNA replication fork progression. Non-B DNA structures - such as hairpins, slipped DNA structures, triplexes, or “sticky” DNA - have been shown to form in vitro at TNR-containing sequences and excellent correlation has been found between the length of the repeated tracts required to adopt such structures and the length found in carriers and patients of the corresponding disease. Nonetheless, compelling evidence is still missing on which structures indeed form in human cells and contribute to TNR instability during DNA replication.
We established a plasmid-based system to recover abundant human replication intermediates and combined gel electrophoresis and electron microscopy to study in vivo fork structure and progression across GAA repeats. We found that replication forks pause transiently and reverse at expanded GAA tracts in both orientations. Furthermore, we identified replication-associated intramolecular junctions involving GAA and other homopurine-homopyrimidine tracts, which we link to pausing and breakage of the sister plasmid fork not traversing the repeats. Finally, we show postreplicative, sister-chromatid hemicatenanes on control plasmids to be converted into persistent homology-driven junctions at expanded GAA repeats (Figure 3). Overall, these data provide novel insight into how premutation GAA tracts interfere with replication and suggest new working hypotheses for trinucleotide repeat expansion. We now plan to combine the powerful investigation system described above with genetic tools (siRNAs), to test the role of candidate mammalian factors in the formation/resolution of the recently identified GAA-specific structures and, more in general, in the stability of repetitive tracts during replication. By large-scale molecular biological approaches, we also recently isolated these abnormal DNA structures and used them to generate specific antibodies, in collaboration with the specialized group of Dr. Mori in Japan. We succeeded in isolating a promising structure-specific antibody and we are currently testing it for in vivo immuno-fluorescence and several in vitro assays. We aim to use this antibody to generate a simple read-out for genomic instability arising at replicating chromosomes.
Figure 3. Expanded GAA/TTC repeats induce unusual replication intermediates in human cells. (A)Neutral-neutral 2D-gel analysis of plasmids containing the indicated numbers of GAA or TTC repeats as template for lagging strand synthesis. Plasmids were transfected in 293T cells, recovered after 40h, digested by EcoRI (A), processed by 2D-gel and probed with the fragment depicted in gray. In the map: circle, SV40 origin; black square, GAA/TTC repeats. Intermediates specific to GAA/TTC repeats are indicated. Black arrow: "2N-spot"; white arrow(s): "Y-spot(s)"; gray circle/rectangle: "1N-spot(s)". (B) Representative electron micrograph of a molecule migrating in the gel area of the 2N spot from GAA90 plasmid EcoRI-fragment. Magnification 46kx.
C. Follonier, J. Oehler, R. Herrador and M. Lopes (2013). Friedreich's Ataxia associated GAA repeats induce replication fork reversal and unusual molecular junctions in human cells. Nature Structural and Molecular Biology, 20: 486–494
C. Follonier and M. Lopes (2013). Combined bi-dimensional electrophoresis and electron microscopy to study specific DNA replication intermediates on human plasmids. In "Functional Analysis of DNA and Chromatin", Humana Press, ed. J. C. Stockert. Methods in Molecular Biology, in press
DNA replication stress in stem cells?
Embryonic stem cells (ESCs) have the unique ability to self-renew and are capable of differentiating into multiple cell types. Therefore, ESCs need to constantly cope with the need to populate any given niche. In contrast, exhaustion of many adult stem cells - haematopoietic stem cells (HSCs) in particular - has been linked to ageing, but the underlying molecular mechanisms are largely unknown. Several knockout-mouse models have uncovered a role for numerous DNA repair factors in ageing and cancer. Besides well-known repair activities, conditional deletion of the ATR gene - which is a central factor activated in response to DNA replication stress - causes depletion of the stem cell niche, suggesting that stem cells need to protect their genome during active proliferation. We recently started to investigate the intriguing connection between replication stress and aging, applying some of our most revealing approaches to different populations of stem cells (ESCs and HSCs). We have found that ESCs exhibit high levels of the endogenous DNA damage marker γH2AX foci, which markedly decreases upon induction of differentiation (Figure 4), when the differentiating cells are still actively dividing. Hence, stemness seems inherently associated with genotoxic stress. Interestingly ESCs lack 53BP1 foci, but exhibit strong staining for RPA and Rad51, suggesting that the observed DDR activation results from perturbations of the replication process, rather than DNA breakage. Similarly, HSCs undergoing replication in basal conditions - or upon interferon a-induced proliferation - show DDR activation and markedly reduced rate of nucleotide incorporation. We now aim to take advantage of our most specialized methods - DNA fiber spreading and electron microscopy - to characterize in more detail this putative replication stress in stem cells. These studies could significantly advance our knowledge on how ESCs proliferate rapidly while maintaining their genome stability; also, they could shed light on the cellular mechanisms leading to stem cell exhaustion in ageing individuals.
M. Lopes, C. Cotta-Ramusino, A. Pellicioli, G. Liberi, P. Plevani, M. Muzi-Falconi, C. S. Newlon and M. Foiani The DNA replication checkpoint response stabilizes stalled replication forks Nature 412, 557-561 (2001)
M.Foiani, A.Pellicioli, M.Lopes, C.Lucca, M.Ferrari, G.Liberi, M.Muzi Falconi, and P.PLevani. DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae. Mutat Res.451(1-2):187-96.(2000)
G. Liberi, Chiolo I., Pellicioli A., Lopes M., Muzi-Falconi M., Plevani P. and Foiani M. Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1- dependent pathway and CDK1 activity. EMBO J. 19, 1, (2000).
A.Pellicioli, C.Lucca, G.Liberi, F.Marini, M.Lopes, P.Plevani, A.Romano, P.Di Fiore, and M.Foiani. (1999) Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase. EMBO J., 18, 6561-6572. (1999)
M.Foiani, M.Ferrari, G.Liberi, M.Lopes, C.Lucca, F.Marini, A.Pellicioli, M.Muzi-Falconi, P.Plevani (1998). S-phase DNA damage checkpoint in budding yeast. Biol.Chem. 379, 1019-1023.