Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Genetic stability in quiescence – quiescenceDNA

Genetic stability in quiescence

How the genetic material is maintained in absence of proliferation is not understood. We want unveils the mutation landscape accumulating during quiescence and study the fate of cells with short telomeres during quiescence

Natural selection in quiescence

Life is maintained through alternating phases of cell division and quiescence over time. During the division the spontneous mutation rates are expressed as the probability of mutants per generation whereas during quiescence the mutation rate can be simply expressed per unit time. We want to determine the mutation rate and spectrum of mutation and the telomere size during quiescence. We will study the role of the layer of protection and DNA damage response in the absence of DNA replication.<br />In absence of telomerase, the telomeres will progressively shorten with each division. This natural erosion of telomeres is due to the normal process of linear DNA replication and eventual replication fork accidents. This phenomenon is called replicative senescence and was proposed to count the number of cell generation. At late time-point of senescence, most of the cells die or remain arrested. Cells eventually recover from growth and establish survivors after 100 generations in fission yeast. Most cells survive by circularizing all chromosomes to bypass the need of telomerase or cells can also survive by maintaining their telomeres, presumably through homologous recombination. We want to study the fate of cells with short telomers during quiescence and their location. Perhaps that in quiescence the telomers are used as molecular clock?

To study the genetic of the quiescence we first isolate from a genetic cross a S. pombe, progenitor reference strain that is haploid and prototrophic. After a short period of growth we remove the nitrogen source. In this conditions the cells divide twice stop in G1 and enter into quiescence. They remain viable for weeks, are metabolically and transcriptionally active as far as glucose and oligoelements are present. Phenotypic mutation rates were determined by plating sample of the same culture after 1, 2, 4, 8 et 16 days in presence of 5-fluorootic acid (5-FOA) that counter select the ura4 and ura5 loss-of-function mutants. The ura4 and ura5 mutants can be latter sequence. We can keep the culture more longer for 1 to 3 months and sequence the whole genome of several individuals to determine the mutation rate and spectrum in the absence of selection. We can latter extend the approach to yeast strains mutated in different DNA repair pathways.
We will study at the molecular and cellular level the telomere during quiescence.

We scored and isolated spontaneous mutations (FOA-resistant) with a rate of 0.6 10-7 events per day of quiescence. The sequencing of the mutants revealed that SNPs accumulate 6 times faster than SNPs on a contrary of what it is observed during proliferation. Conversion of the phenotypic mutation rate indicates that 3 months of quiescence are necessary to generate few mutations per genome. Illumina library have been constructed and sequenced (paired-end and 50X coverage). Over 30 cells we found in average 0.8 SNP and 1.3 indels per genome of 3 month age, with 80% and 70% FDR, respectively. A manuscript is in preparation. We found that the uracile DNA repair pathway is required during quiescence. An biochemical study is underway.
In collaboration with Sylvie Tournier (Toulouse) we observed that the telomeres progressively relocated at the rDNA. We observed that short telomeres are rearranged during quiescence. The appearance of rearranged telomeres is correlated with the shortening of telomeres and the time spent in G0. Telomere rearrangements that we observed in quiescence are different from those described during senescence. We confirmed that chromosomes are not circularized and that telomeric sequences are not maintained. Instead, we found that these rearrangements correspond to the amplification of subtelomeric regions through homologous recombination. However, they are not stable when cells return to growth.

Considering that proliferation as well as quiescence is under natural selection, a direct extension of the above results is that each gamete alternatively selects for high performance of proliferation or quiescence, those qualities ultimately merge in the diploid zygote, prior to the initiation of a new life cycle. If this is correct, even partially, it suggests that the fundamental difference of the two gamete life-styles contribute to the genetic performances of the somatic cells and the homeostasis of adult tissues with ageing, We reveal a novel telomeric organization during quiescence that is apparently not compatible with growth.

One manuscript is under preparation and oral presentaitons at meeting have been done.

The two greatest threats to human health in developed countries are cancer and degenerative diseases. Numerous studies have indicated that cancer cells show increased genomic instability related to defective DNA repair and response to DNA damage during proliferation. Genomic instability has been also implicated in several neurodegenerative disorders including amyotrophic lateral sclerosis and ataxia. It has been proposed that the highly metabolically active neurons are particularly vulnerable to DNA damage and that their post-mitotic lifespan relies on specific DNA surveillance and repair systems. This raises the question how DNA damage is repaired in post-mitotic non-dividing cells.

Pioneer works from Mitsuhiro Yanagida’s laboratory have shown that the fission yeast Schizosaccharomyces pombe is a great model for cellular quiescence. S. pombe can be experimentally maintain for weeks in quiescence in the absence of nitrogen. Published and preliminary results from this consortium revealed important new findings on the mechanisms required for genetic stability and cell viability in the absence of cell division. In this research project we want to focus on some long-standing questions: What is the hierarchy of the various DNA repair pathways to infer on the physiological sources of lesions? How unstable sequences are processed in quiescence? How telomeres are maintained during quiescence?

To address these questions we will coordinate the effort of two laboratories specialized in chromatin, DNA repair and telomeres and use the advanced molecular and cellular genetics tools and approaches available for S. pombe. The project will be divided in several main tasks. We will first study the viability in quiescence of the wild type and DNA repair mutant strains and determine their mutational spectrum using counter-selectable markers or whole genome sequencing. Second, we aim to understand the mechanisms responsible of the instability and processing of an expanded hexanucleotide GGGGCC repeat involved in the lateral sclerosis (ALS) and fronto-temporal dementia (FTD). Finally, we will intend to better understand the maintenance of telomeres in quiescent cells in the presence and absence of telomerase activity and the possible crosstalk between telomere length and the DNA damage response in fission yeast. We anticipate disclosing the mechanisms involved in the maintenance of genome integrity of quiescent cells and we believe that this knowledge might have a profound impact on our understanding of age-related diseases.

Project coordination

Benoit ARCANGIOLI (Unité de la Dynamique du génome, Institut Pasteur)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Institut Pasteur Unité de la Dynamique du génome, Institut Pasteur
CNRS DR12_CRCM Centre national de la Recherche Scientifique Délégation Provence et Corse _ Centre de recherche en cancérologie de Marseille

Help of the ANR 379,916 euros
Beginning and duration of the scientific project: December 2013 - 36 Months

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