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Nearly half the human genome is covered with repetitive DNA sequences. Whilst being important for gene expression, these genomic loci are also intrinsically difficult to replicate during cell division, and therefore represent hotspots for genomic rearrangements frequently promoting carcinogenesis and other human diseases. Additionally, active transcription as needed for gene expression can compete with ongoing replication for the same DNA template – a conflict requiring tremendous spatiotemporal coordination in order to protect the cell’s integrity. It has recently become increasingly clear that these transcription-replication conflicts (TRCs) have an early causative role in genome instability and tumorigenesis, but the underlying molecular mechanisms and DNA intermediates remain yet to be discovered, giving rise to a growing number of speculative models.
Main goal of this research line is to clarify how specific DNA sequence or interference with other aspects of DNA metabolism challenge the DNA replication process, elucidating cellular mechanisms limiting endogenous replication stress and protecting the integrity of replicating chromosomes.
Our lab has taken advantage of episomal DNA replication systems, molecular biology approaches and electron microscopic analysis of replication intermediates to visualise directly in human cells how replication forks are challenged by repetitive DNA sequences and to understand how cells limit their instability during DNA synthesis. We recently focused on unravelling key molecular mechanisms occurring at TRCs, often associated with RNA-DNA hybrid accumulation, and established several approaches to monitor key intermediates associated with transcription-replication interference. We now plan to exploit this experimental platform to investigate whether and how TRCs underlie genomic instability and tumorigenesis in specific cell types, particularly exposed to this peculiar type of replication stress.
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 Struct Mol Biol, 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.
A. Ray Chaudhuri, A. K. Ahuja, R. Herrador and M. Lopes (2015). PARG prevents the accumulation of unusual replication structures during unperturbed S phase. Mol Cell Biol. 35:856-65
R. Zellweger and M. Lopes (2018). Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy. In "Genome Instability". Humana Press, ed. G. Brown and M. Muzi Falconi. Methods in Molecular Biology, 1672:261-294.
J.A. Schmid, M. Berti, F. Walser, M.C. Raso, F. Schmid, J. Krietsch, H. Stoy, K. Zwicky, S. Ursich, R. Freire, M. Lopes° and L. Penengo° (2018). Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase. Molecular Cell, 71(6):897-910.e8. doi: 10.1016/j.molcel.2018.07.011. °corresponding authors
H. Stoy, K. Zwicky, D. Kuster, K.S. Lang, J. Krietsch, M. P. Crossley, J. A. Schmid, K. A. Cimprich, H. Merrikh and M. Lopes (2023). Direct visualization of transcription-replication conflicts reveals post-replicative DNA:RNA hybrids. Nature Struct Mol Biol, doi: 10.1038/s41594-023-00928-6
