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  Director : Arcangioli Benoit (barcan@pasteur.fr)



We identified the cis-acting elements responsible for the imprint at the mating-type locus in the fission yeast, Schizosaccharomyces pombe. The molecular nature of the imprint has been determined as a single-strand break, and we constructed and validated a inducible mating-type switching system. We showed that Pli1 encodes for a SUMO E3-ligase, which plays an important role for chromosome stability.



Our previous works allowed us to identify a single stranded lesion at the mating-type locus mat1, in the fission yeast. This DNA lesion is the initial event triggering mating-type switching in this yeast. Polar replication pausing and termination at mat1 is essential for the formation of the lesion (Dalgaard and Klar, 1999).

Molecular nature of the imprint Atanas Kaykov

Atanas Kaykov performed a linker scanning mutagenesis of the DNA sequence flanking the imprinted site (100 bp) and found three novel cis-acting elements. One element is essential for a replication fork pause and interacts in vivo with the Swi1 protein. The two other sites are important to stabilize the break following its formation (Kaykov et al. 2004).

The molecular nature of the DNA lesion is still controversial. Another team proposed that the imprint is a heat- and alkali-labile modification and suggested that one or two ribonucleotides have been incorporated at mat1. We have challenged this hypothesis and proposed that the lesion is a site-specific single-strand nick, with 3'OH and 5'OH termini, with no nucleotides missing and is resistant to RNases (Kaykov and Arcangioli, 2004).

An inducible single-strand break. Allyson Holmes, Xiaowei Sun

Recently, Allyson Holmes constructed and validated a powerful inducible nick and mating-type switching system. Starting with a homogenous cell population (one mating-type and no break) we were able to study the kinetics of each cellular step. We found that the break appears during S-phase and on the replicating mat1 DNA intermediates during the first replication. The binding of Swi1 is concomitant with the first DNA replication and accumulates until the beginning of the G2-phase and rapidly disappears. As previously inferred, the break is maintained until the following S phase, triggering recombination and mating-type switching. This formal molecular demonstration has been confirmed at the single cell level, by pedigree analysis. The overall process of MT switching in S. pombe is a two-step process, involving two rounds of DNA replication (Holmes at al., 2005).

Coupling recombination to réplication. Laura Roseaulin

The role of recombination in the recovery of stalled/collapsed replication forks is an essential process in maintaining genomic stability and defects in this pathway cause predisposition to many forms of diseases. The situation becomes critical when the replication fork collides with an unrepaired single-strand break, and converts it into a one-ended double-strand break. The repair process must restore a fork structure that is suitable to restart replication. We show that the single collapsed replication fork at the mating-type locus required the homologous recombination enzymes for viability in fission yeast. The molecular intermediates appearing during the repair were analyzed.

Role of Tdp1 in G0. Samia Ben Hassine

Spinocerebellar ataxia (SCAN1) is a neurodegenerative disease that result from mutation of tyrosyl phosphodiesterase 1 (Tdp1). We have shown that Tdp1 from S. pombe is required for the reparation of some single-strand lesions. Interestingly, in the absence of Tdp1, S. pombe cannot survive in G0, a phenotype comparable to the loss of viability in post-mitotic neurons. We are searching for the reasons on this sensibility.

Implication of Pli1, a SUMO E3 ligase in genomic stability. Blerta Xhemalce

SUMO is covalently conjugated to certain proteins, in a similar multistep process to ubiquitination, and seems to direct their targets to particular cellular locations/structures. Blerta Xhemalce in collaboration with J. Seeler (Unité de l'Organisation Nucléaire et Oncogénèse), have shown that Pli1 is an E3 SUMO-ligase in vivo and in vitro. Our genetic studies indicated that Pli1 and SUMO modification plays an important role in the protection of repetitive elements within heterochromatin from illegitimate homologous recombination, especially at the mating-type, centromere and telomeric region. We have shown that the telomeric repeat extension, in the absence of Pli1, is due to an increase activity of the telomerase.

Keywords: Imprinting, recombination, replication, repair, cancer


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