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Recombination and Genetic Instability

Keywords : recombination, DNA repair, DNA replication, meiosis, yeast

Group leader : Alain Nicolas

 

All cellular DNA is liable to damage, which can involve single-strand breaks, double-strand breaks or alterations to the nucleotide base structure. Such damage may arise spontaneously, it may result from programmed recombination events (as in meiosis, for example) or it may be induced by environmental factors like radiation or chemical mutagens. In all cases, these lesions must be repaired to conserve the integrity of the genetic material for future generations of cells, to prevent possible formation of cancers, and, in the short-term, to allow the damaged cell to survive. Usually, most DNA damage is repaired accurately, but defects in the repair systems may allow anything from single-base alterations to large-scale rearrangements to persist.

We are studying two different situations in which DNA double-strand breaks form in the budding yeast Saccharomyces cerevisiae: the first, during meiosis when sister chromosomes exchange genetic material by recombination and, the second, the instability of human tandem repeated DNA sequences (minisatellites) inserted in the yeast genome.

Early in meiotic recombination, Spo11, a meiosis-specific endonuclease, induces the DNA double-strand breaks that initiate exchange of genetic material between the homologous chromosomes. Repair of these double-strand breaks leads to crossover exchanges between non-sister chromatids and thus ensures segregation of the recombined chromosomes at the first meiotic division.

To map where these double-strand breaks occur throughout the entire genome, we used the combined methods of chromatin immunoprecipitation (ChIP) and microarrrays (chip); we discovered that each chromosome has a unique map, with alternating ‘hot' and ‘cold' domains where recombination occurs more or less frequently (Fig. 1).

Fig.1: Genome-wide 'ChIP-chip' map of the DNA double-strand breaks induced by Spo11 (left panel) and of the Mre11 protein fixing (right panel).Fig.1: Genome-wide 'ChIP-chip' map of the DNA double-strand breaks induced by Spo11 (left panel) and of the Mre11 protein fixing (right panel).

The factors controlling formation of meiotic double-strand breaks are not well known. In S. cerevisiae, at least 10 genes are required besides SPO11. To identify the roles of the proteins they encode, we studied the association of these proteins with chromatin during meiosis by ChIP-chip analysis. When compared with the Spo11 cleavage map, we see that Mre11 (Fig. 2), as well as Rad50 and Xrs2 (called Nbs1 in mammals), preferentially associate with the double-strand breaks sites.

Fig.2: Time-course of double-strand break formation in the GAL2 gene upon induction of meiosis. The natural protein Spo11 does not form double-strand breaks in the 5' region of the GAL2 gene (left panel). The Gal4BD-Spo11 fusion protein, by contrast, produces a testFig.2: Time-course of double-strand break formation in the GAL2 gene upon induction of meiosis. The natural protein Spo11 does not form double-strand breaks in the 5' region of the GAL2 gene (left panel). The Gal4BD-Spo11 fusion protein, by contrast, produces a test

To modify the sites of DNA cleavage by Spo11, we fused it to the DNA-binding domain of the protein Gal4. This fusion protein introduces double-strand breaks not only in the ‘natural' recombination regions, but also in chromosomal regions that are normally refractory (Fig. 2). Since Spo11 is present throughout eukarya, we plan to apply this method to study meiotic homologous recombination in other species too.

In the S. cerevisiae genome, as in the human genome, tandem repeated minisatellite DNA sequences are unstable during meiosis when they may undergo expansion and/or contraction of the number of tandem repeats. To investigate the mechanism(s) underlying tandem-repeat instability, we introduced two human minisatellite CEB1 alleles into the S. cerevisiae genome. We found that deletion of the RAD27 gene, which is involved in DNA replication and repair (Fig. 3), causes a high level of instability of the CEB1-1.8 allele in cells growing vegetatively, indicating that replication defects destabilise these repeated sequences. It gives rise to a large variety of length variants due to expansion and contraction of the repeat units (Fig. 4).

Fig.3: The mode of action of Rad27/FEN1. Rad27/FEN1 is a Flap endonuclease that cleaves the Okasaki fragments made during DNA replication.Fig.3: The mode of action of Rad27/FEN1. Rad27/FEN1 is a Flap endonuclease that cleaves the Okasaki fragments made during DNA replication.Fig.4: Examples of rearrangements of the minisatellite sequence CEB1-1.8 found in the S. cerevisiae strain rad27D.Fig.4: Examples of rearrangements of the minisatellite sequence CEB1-1.8 found in the S. cerevisiae strain rad27D.

Our current projects aim to characterise further the normal functions of Rad27 and its human orthologue, FEN1, as well as to study the pathological consequences of mutations in these proteins in yeast and human cells.