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Characterizing the role of the Est1 protein in stimulating the recruitment of the Est3 protein to the yeast telomerase complex

dc.creatorRiddle, Abigail Leigh
dc.description.abstractTelomeres are protective protein/DNA complexes that cap the ends of linear chromosomes. The repetitive, TG-rich telomeric sequences are elongated by the telomerase ribonucleoprotein. In Saccharomyces cerevisiae, the catalytic core of telomerase consists of the reverse transcriptase Est2p and the TLC1 RNA, which contains the template for nucleotide addition. The Est1 and Est3 proteins serve regulatory roles in vivo but are dispensable for in vitro telomerase activity. The mechanism through which Est3p assembles with the holoenzyme is debated within the field, specifically concerning Est1p’s role in Est3p recruitment. Based on evidence demonstrating that Est1p is not absolutely required to recruit Est3p, I hypothesize that Est1p stimulates Est3p recruitment. To identify residues of the Est1 protein responsible for this function, I used a combination of in vivo and in vitro experiments and two genetic screens. Using an in vivo assembly assay, I determined that a putative Est3p recruitment domain lies between amino acids 499 and 563 of Est1p and optimized the protocol for analyzing point mutations within this region. Additionally, I have designed two genetic screens to isolate alleles of EST1 specifically disrupted for stimulating Est3p recruitment. The first uses an Est2-Est3 fusion protein predicted to bypass only the Est3p recruitment function of Est1p. The second utilizes a galactose-inducible allele of EST3 to screen for Est1p mutants rescued by over-expression of Est3p. Identification of separation-of-function alleles of EST1 with these tools will clarify the precise mechanisms of yeast telomerase complex assembly and potentially provide insight into the interactions of human telomerase components.
dc.subjectDNA replication
dc.subjectprotein complex
dc.titleCharacterizing the role of the Est1 protein in stimulating the recruitment of the Est3 protein to the yeast telomerase complex
dc.contributor.committeeMemberKatherine Friedman
dc.type.materialtext Sciences University
dc.contributor.committeeChairBrandt Eichman

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