Unit: Genome Dynamics
Director: Arcangioli Benoit
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
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).
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.
Keywords: replication, recombination, chromatin, cancer