A yeast model of Bloom's syndrome.

Chakraverty, Ronjon (1999). A yeast model of Bloom's syndrome. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00010208


Bloom's syndrome (BS) is a rare inherited disorder associated with growth retardation, immunodeficiency and a predisposition to cancers of all types. At a cellular level BS is associated with marked chromosomal instability, as manifest by interchanges between homologous chromosomes and sister chromatids. BS arises as a consequence of defects in a DNA helicase, BLM, which is a member of the RecQ family of helicases. Defects in other RecQ family helicases, derived from prokaryotic and lower eukaryotic cells, also result in a loss of genomic integrity. This suggests that this group of helicase subfamily share a degree of functional conservation.

In this thesis, the validity of using the budding yeast, S.cerevisiae, as a model system for BS has been assessed. In this organism, the structural homologue of the BLM protein is Sgs1p. Deletion of Sgs1p is associated with mitotic hyperrecombination and defective chromosome segregation. Deletion of SGS1 also suppresses the slow growth and hyperrecombination phenotype of top3 mutants, which are defective in topoisomerase III. Ectopic expression of a wild type SGS1 gene in sgs1Δtop3Δ mutants reinduced the slow growth defect, and this effect was reproduced when the human BLM gene was expressed. This result suggested firstly, that the yeast and human proteins share functional similarities and secondly, that the interaction between the RecQ family helicases and topoisomerase III is highly conserved. On this basis, the remainder of this thesis focussed upon the role of topoisomerase III.

Deletion of the S.cerevisiae TOP3 gene leads to a slow growth phenotype accompanied by an accumulation of cells with a late S/G2 content of DNA. top3Δ mutants exhibit a RAD24/RAD9-dependent delay in the G2 phase, suggesting a role for topoisomerase III in the resolution of abnormal DNA structures/damage arising during S-phase. Consistent with this notion, top3Δ strains are defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA damaging agents and are sensitive to killing by a variety of DNA damaging agents, including ultra-violet light, ɣ-rays and the alkylating agent MMS. This S-phase checkpoint defect was not associated with a failure to induce expression of DNA damage response genes. Consistent with an S-phase specific role for topoisomerase III, expression of the TOP3 mRNA is activated in the late G1 phase, and DNA damage checkpoints operating outside of S-phase are unaffected by deletion of TOP3. All of the phenotypic consequences of loss of topoisomerase III function were suppressed by deletion of SGS1. These data implicate topoisomerase III and, by inference, Sgs1p in a checkpoint role in response to DNA damage arising during S-phase. A model to explain the role of these proteins during DNA replication is proposed.

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