Browsing by Author "Brosnan, Laura"

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    Regulation of Human HRAS1 Minisatellite Stability During Stationary Phase
    (2009-10-07) Brosnan, Laura
    Minisatellites are repetitive tracts of DNA with repeat units ranging from 16-100 base pairs in length. They are stable during mitosis but display changes in repeat number and order after meiosis. Rare alleles of minisatellite tracts thought to arise from repeat instability are associated with human diseases, including cancer, diabetes, and epilepsy. The stability of minisatellites in non-proliferating stationary phase cells is not well understood. Previous work has shown that the zinc transporter ZRT1, the checkpoint gene RAD53, the DNA repair gene RAD27, the endocytosis gene END3, and the protein kinase PKC1 regulate the stability of minisatellites in S. cerevisiae during stationary phase. We inserted the human minisatellite associated with HRAS1 into the ADE2 gene to determine how its stability is regulated during stationary phase. Loss of ZRT1, RAD27, or RAD53 destabilized the minisatellite; loss of PKC1 or END3 had no effect. This work contributes significantly to our understanding of repeat stability and genome stability during stationary phase; this has important implications for human genome stability, since most human somatic cells are non-proliferating.
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    Regulation of Human HRASI Minisatellite Stability During Stationary Phase
    (2011-12-14) Brosnan, Laura;
    The majority of eukaryotic cells exist in a quiescent state outside of the cell cycle known as stationary phase, or G0. Although quiescent cells are non-proliferating, they retain the ability to reenter the cell cycle. Events such as DNA alterations can take place in stationary phase cells that are associated with a risk for inappropriate reentry into the cell cycle and the development of cancer. Such alterations can occur in repetitive tracts of DNA known as minisatellites. When change in the number or order of minisatellite repeat units occurs, rare alleles of minisatellite tracts may arise. Many of these rare alleles are associated with human diseases, including cancer, diabetes, and epilepsy. Previous work in our lab has demonstrated that certain genes are involved in regulating the stability of a synthetic minisatellite in stationary phase cells. In order to relate these findings to the association between minisatellites and human disease, we inserted the human minisatellite associated with the HRAS1 oncogene into the ADE2 gene of S. cerevisiae to determine how its stability is regulated during stationary phase. Our lab has developed a novel assay for studying stationary phase minisatellite stability in S. cerevisiae. When minisatellites are destabilized and undergo a change in repeat number or order, a novel color phenotype known as blebbing occurs. Blebbing mutants were constructed and we found that the zinc transporter ZRT1, the DNA repair gene RAD27, the checkpoint gene RAD53, the Polε subunit DPB3, and several checkpoint genes (including MRC1, TOF1, CSM3) regulate the stability of the human HRAS1 minisatellite. Upon examination of minisatellite alterations in these blebbing mutants, we saw both expansions and contractions of the minisatellite tract; through further analysis, these alterations were found to be dependent upon homologous recombination.