Photo: Herrador, Vujanovic, Terraneo, Zwicky, Mijic, Dalcher, Lopes, Ahuja, Mutreja
|Group Leader||Massimo Lopes|
|PhD Students||Sofija Mijic, Karun Mutreja, Marko Vujanovic, Katharina Zwicky|
|Technical Assistants||Raquel Herrador, Ralph Zellweger|
|Master Student||Nastassja Terraneo|
|Postdocs||Arnab Ray Chaudhuri, Cindy Follonier, Kai Neelsen|
|Master Students||Isabella Zanini, Judith Oehler|
|Civil Servant||Damian Dalcher|
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, which contribute to 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 fiber spreading, psoralen-crosslinking coupled to electron microscopy).
Structural insights into oncogene-induced DNA replication stress
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. 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 about the DNA structures generated in vivo under such conditions.
Replication stress phenotype can be reproduced in cell culture by overexpression of various oncogenes influencing DNA replication, e.g. Cyclin E or 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 oncogene induction. However, only the overexpression of Cdc25A 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 arise due to premature activation of mitotic factors. Using a similar set of approaches we have also recently characterized the molecular consequences of "re-replication", a deregulation of a replication initiation program that is frequently associated with tumorigenesis. We are now in the process of extending our studies to a broad spectrum of oncogenes, in an attempt to identify common molecular mechanisms underlying tumorigenesis. Furthermore, we will test the involvement of known cancer susceptibility factors in these molecular processes, as we suspect that altered cellular responses to replication stress could underlie the high incidence of cancer associated with certain genetic defects.
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, indicative of active DNA damage response. (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, I.M.Y. Zanini, S. Mijic, R. Herrador, R. Zellweger, A. Ray Chaudhuri, K.D. Creavin, J.J. Blow and M. Lopes(2013). Deregulated origin licensing leads to chromosomal breaks by re-replication of a gapped DNA template. Genes and Development, 27:2537-42
K. J. Neelsen, A. Ray Chaudhuri, C. Follonier, R. Herrador andM. Lopes(2014). 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, 1094:177-208.
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 clinic 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. We now plan to test the contribution of specific cellular factors likely to play a role in the 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 restart 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 the 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 Syndrome 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 showed that TNR can cause pausing of DNA replication forks. 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 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 insights 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.
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 (2014). 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, 1094:209-19.
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 genomes during active proliferation. We recently started to investigate the intriguing connection between replication stress and ageing in different populations of stem cells (ESCs and HSCs) by a variety of techniques available in the laboratory. We found that ESCs contain numerous sub-nuclear foci of the endogenous DNA damage marker γH2AX, which markedly diminish 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 under standard 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 of 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.
A powerful technological platform to support structural DNA investigations worldwide
Thanks to the rare combination of specialized approaches, our laboratory has also been very actively involved in worldwide collaborative efforts requiring structural insights into DNA metabolism. A number of projects have been published in high-impact journals, while several other collaborative efforts are either ongoing or approaching the publication stage.
J. Mejlvang, Y. Feng, C. Alabert, K.J. Neelsen, Z. Jasencakova, X. Zhao, M. Lees, A. Sandelin, P. Pasero, M. Lopes and A. Groth (2014).New histone supply regulates replication fork speed and PCNA unloading. Journal of Cell Biology, 204: 29-43
J. Peńa-Diaz, S. Felscher, C. Follonier, D. Castor, M. Lopes, A. A. Sartori and J. Jiricny (2012). Noncanonical Mismatch Repair as a Source of Genomic Instability in Human Cells. Mol Cell, 47:669-80
I. M. Toller*, K.J. Neelsen*, M. Steger, M. O. Hottiger, M. Gerhard, A. A. Sartori, M. Lopes° and A. Müller° (2011). The carcinogenic bacterial pathogen Helicobacter pylori triggers DNA double strand breaks and a DNA damage response in infected host cells. PNAS 108:14944-9 *equal contribution °corresponding authors
K. Engels, M. Giannattasio, M. Muzi-Falconi, M. Lopes° and S. Ferrari° (2011). 14-3-3 Proteins Regulate Exonuclease 1-Dependent Processing Of Stalled Replication Forks. PLoS Genetics, 7:e1001367 ° corresponding authors
Y. Hashimoto, A. Ray Chaudhuri, M. Lopes° and V. Costanzo°. (2010) Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nature Struct Mol Biol, 17:1305-131 ° corresponding authors
M. Giannattasio, C. Follonier, H. Tourričre, F. Puddu, F. Lazzaro, P. Pasero, M. Lopes, P. Plevani and M. Muzi-Falconi. (2010) Exo1 competes with repair synthesis, converts NER intermediates to long ssDNAgaps and promotes checkpoint activation. Mol Cell, 40:50-62
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.