Publications

2022
Artemis G. Korovesi, Leonor Morgado, Marco Fumasoni, Ricardo Henriques, Hannah Heil, and Mario Del Rosario. 3/8/2022. “Expansion Microscopy on Saccharomyces cerevisiae”. Publisher's VersionAbstract
he unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross- linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.
Korovesi_et_al_2022.pdf
Valeria Dolce, Sabrina Dusi, Michele Giannattasio, Chinnu Rose Joseph, Marco Fumasoni, and Dana Branzei. 2/3/2022. “Parental histone deposition on the replicated strands promotes error-free DNA damage tolerance and regulates drug resistance.” Genes & Development, 36, Pp. 1-13. Publisher's VersionAbstract
Ctf4 is a conserved replisome component with multiple roles in DNA metabolism. To investigate connections between Ctf4-mediated processes involved in drug resistance, we conducted a suppressor screen of ctf4Δ sensitivity to the methylating agent MMS. We uncovered that mutations in Dpb3 and Dpb4 components of polymerase ε result in the development of drug resistance in ctf4Δ via their histone-binding function. Alleviated sensitivity to MMS of the double mutants was not associated with rescue of ctf4Δ defects in sister chromatid cohesion, replication fork architecture, or template switching, which ensures error-free replication in the presence of genotoxic stress. Strikingly, the improved viability depended on translesion synthesis (TLS) polymerase-mediated mutagenesis, which was drastically increased in ctf4 dpb3 double mutants. Importantly, mutations in Mcm2–Ctf4–Polα and Dpb3–Dpb4 axes of parental (H3–H4)2 deposition on lagging and leading strands invariably resulted in reduced error-free DNA damage tolerance through gap filling by template switch recombination. Overall, we uncovered a chromatin-based drug resistance mechanism in which defects in parental histone transfer after replication fork passage impair error-free recombination bypass and lead to up-regulation of TLS-mediated mutagenesis and drug resistance.
2021
Marco Fumasoni and Andrew W. Murray. 11/9/2021. “Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress.” PLoS Genetics, Pp. 10.1101/2020.12.17.423282. Publisher's VersionAbstract

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules despite differences in genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

2020
Marco Fumasoni. 10/27/2020. “Tell Me Where You’ve Been and I’ll Tell You How You’ll Evolve.” mBio, 11:e02043-20. Publisher's VersionAbstract
The reproducibility of adaptive evolution is a long-standing debate in evolutionary biology. Kempher et al. (M. L. Kempher, X. Tao, R. Song, B. Wu, et al., mBio 11:e00569-20, 2020, https://doi.org/10.1128/mBio.00569-20) used experimental evolution to investigate the effect of previous evolutionary trajectories on the ability of microbial populations to adapt to high temperatures. Despite the divergence caused by adaptation to previous environments, all populations reproducibly converged on similar final levels of fitness. Nevertheless, the genetic basis of adaptation depended on past selection experiments, reinforcing the idea that previous adaptation can dictate the trajectories of later evolutionary processes.
fumasoni_2020.pdf
Madhusudhan Srinivasan, Marco Fumasoni, Naomi J Petela, Andrew W Murray, and Kim A Nasmyth. 6/9/2020. “Cohesion is established during DNA replication utilising chromosome associated cohesin rings as well as those loaded de novo onto nascent DNAs.” eLife, eLife 2020;9:e56611. Publisher's Version srinivasan_et_al_2020.pdf
Thomas LaBar, Yu-Ying Phoebe Hsieh, Marco Fumasoni, and Andrew W. Murray. 5/18/2020. “Evolutionary repair experiments as a window to the molecular diversity of life.” Current Biology. Publisher's VersionAbstract
Comparative genomics reveals an unexpected diversity in the molecular mechanisms underlying conserved cellular functions, such as DNA replication and cytokinesis. However, the genetic bases and evolutionary processes underlying this ‘molecular diversity’ remain to be explained. Here, we review a tool to generate alternative mechanisms for conserved cellular functions and test hypotheses concerning the generation of molecular diversity —evolutionary repair experiments, in which laboratory microbial populations adapt in response to a genetic perturbation. We summarize the insights gained from evolutionary repair experiments, the spectrum and dynamics of compensatory mutations, and the alternative molecular mechanisms used to repair perturbed cellular functions. We relate these experiments to the modifications of conserved functions that have occurred outside the laboratory. We propose experimental strategies, especially those that establish a quantitative understanding of compensatory mutations and alternative molecular mechanisms, to improve evolutionary repair as a tool to explore the molecular diversity of life.
Marco Fumasoni and Andrew W. Murray. 2/11/2020. “The evolutionary plasticity of chromosome metabolism allows adaptation to constitutive DNA replication stress.” eLife, Pp. eLife 2020;9:e51963. Publisher's VersionAbstract
Many biological features are conserved and thus considered to be resistant to evolutionary change. While rapid genetic adaptation following the removal of conserved genes has been observed, we often lack a mechanistic understanding of how adaptation happens. We used the budding yeast, Saccharomyces cerevisiae, to investigate the evolutionary plasticity of chromosome metabolism, a network of evolutionary conserved modules. We experimentally evolved cells constitutively experiencing DNA replication stress caused by the absence of Ctf4, a protein that coordinates the enzymatic activities at replication forks. Parallel populations adapted to replication stress, over 1000 generations, by acquiring multiple, concerted mutations. These mutations altered conserved features of two chromosome metabolism modules, DNA replication and sister chromatid cohesion, and inactivated a third, the DNA damage checkpoint. The selected mutations define a functionally reproducible evolutionary trajectory. We suggest that the evolutionary plasticity of chromosome metabolism has implications for genome evolution in natural populations and cancer.
fumasoni_and_murray_2020.pdf
2015
Marco Fumasoni, Katharina Zwicky, Fabio Vanoli, Massimo Lopes, and Dana Branzei. 3/5/2015. “Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.” Molecular Cell, 57, 5, Pp. 812-823. Publisher's VersionAbstract
Chromosomal replication is entwined with DNA damage tolerance (DDT) and chromatin structure establishment via elusive mechanisms. Here we examined how specific replication conditions affecting replisome architecture and repriming impact on DDT. We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion. The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing. Notably, the DDT defects of Polα/Primase/Ctf4 mutants are not the consequence of increased sister chromatid distance, but are instead caused by altered single-stranded DNA metabolism and abnormal replication fork topology. We propose that error-free TS is driven by timely replicative helicase-coupled re-priming. Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.
Fumasoni_et_al_2015.pdf
2014
Victor Gonzalez‐Huici, Barnabas Szakal, Madhusoodanan Urulangodi, Ivan Psakhye, Federica Castellucci, Demis Menolfi, Eerappa Rajakumara, Marco Fumasoni, Rodrigo Bermejo, Stefan Jentsch, and Dana Branzei. 1/31/2014. “DNA bending facilitates the error‐free DNA damage tolerance pathway and upholds genome integrity.” The EMBO journal, 33, 4, Pp. 327-340. Publisher's VersionAbstract
DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination‐mediated (error‐free) and translesion synthesis‐mediated (error‐prone) DNA damage tolerance pathways. Crucial for error‐free DNA damage tolerance is template switching, which depends on the formation and resolution of damage‐bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication‐associated lesions into the error‐free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C‐terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication‐associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.
Gonzalez-huici_et_al_2014.pdf
2013
Georgios I. Karras, Marco Fumasoni, Grzegorz Sienski, Fabio Vanoli, Dana Branzei, and Stefan Jentsch. 2/7/2013. “Noncanonical role of the 9-1-1 clamp in the error-free DNA damage tolerance pathway.” Molecular Cell, 43, 3, Pp. 536-546. Publisher's VersionAbstract
Damaged DNA is an obstacle during DNA replication and a cause of genome instability and cancer. To bypass this problem, eukaryotes activate DNA damage tolerance (DDT) pathways that involve ubiquitylation of the DNA polymerase clamp proliferating cell nuclear antigen (PCNA). Monoubiquitylation of PCNA mediates an error-prone pathway by recruiting translesion polymerases, whereas polyubiquitylation activates an error-free pathway that utilizes undamaged sister chromatids as templates. The error-free pathway involves recombination-related mechanisms; however, the factors that act along with polyubiquitylated PCNA remain largely unknown. Here we report that the PCNA-related 9-1-1 complex, which is typically linked to checkpoint signaling, participates together with Exo1 nuclease in error-free DDT. Notably, 9-1-1 promotes template switching in a manner that is distinct from its canonical checkpoint functions and uncoupled from the replication fork. Our findings thus reveal unexpected cooperation in the error-free pathway between the two related clamps and indicate that 9-1-1 plays a broader role in the DNA damage response than previously assumed.
Karras_et_al_2013.pdf
2010
Fabio Vanoli, Marco Fumasoni, Barnabas Szakal, Laurent Maloisel, and Dana Branzei. 11/11/2010. “Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch.” PLoS genetics, 6, 11. Publisher's VersionAbstract
Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair.
Vanoli_et_al_2010.pdf
2009
Kara A Bernstein, Erika Shor, Ivana Sunjevaric, Marco Fumasoni, Rebecca C Burgess, Marco Foiani, Dana Branzei, and Rodney Rothstein. 4/8/2009. “Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair.” The EMBO journal, 28, 7, Pp. 915-925. Publisher's VersionAbstract
Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.