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Functional divergence between Tetrahymena telomere proteins: Potential role for POT1b in chromosome breakage and new telomere synthesis

Heyse, Serena R.

Abstract Details

2011, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.

Telomeres are nucleoprotein complexes that protect chromosomes from degradation and end-to-end fusions, and serve as a substrate for telomerase. The DNA consists of a duplex of repeated sequence followed by a short, single-stranded overhang. The overhang is bound by the protein POT1. Tetrahymena thermophila has two POT1 homologs, POT1a and POT1b. POT1a is essential because it is required to prevent the overhang from eliciting a DNA damage response. It also has a role in telomere length regulation. The function of POT1b is distinct from POT1a. We found that disruption of POT1b does not affect telomere length, overhang structure, or growth rate in vegetative cells. However, RT-PCR analysis revealed that POT1b is developmentally regulated. POT1b expression is upregulated during conjugation at the time the new macronucleus is found and the parental chromosomes are fragmented and new telomeres are added onto each new chromosome end. Immunofluorescence data indicate that POT1b localizes to the developing macronucleus at the time of telomere synthesis, suggesting that POT1b may play a role in the production of new telomeres. To determine whether POT1a or POT1b can bind directly to telomeric DNA, I expressed the DNA-binding domain (DBD) and tested binding to telomeric DNA in a mobility shift gel. Only the DBD of POT1a was able to bind. However, chromatin immunoprecipitation (ChIP) experiments indicate that POT1b interacts with the region of chromosome breakage where new telomeres will be added following cleavage. ChIP also indicates a small enrichment of POT1b at telomeres following the timing of chromosome breakage. This suggests POT1b may be in a protein complex that remains associated with the sites of new telomere synthesis.

In order to further understand the mechanism of telomere synthesis following chromosome breakage, I have begun to compare the chromosome breakage and internal eliminated sequence (IES) excision pathways. These forms of chromosome processing occur late in conjugation as the new macronucleus develops. While it is known that the IES excision pathway occurs by an RNAi like mechanism, little is known about the chromosome breakage pathway. By using a PCR assay to look for the appearance of a chromosome breakage product, I found that chromosome breakage occurs after the cells separate from conjugation at a time comparable to what is seen with IES excision. This assay was also used to determine whether two proteins involved in IES excision, Pdd1 and Dcl1, have a role in chromosome breakage. I found that loss of Pdd1 during early conjugation had no affect on chromosome breakage; whereas, loss of Pdd1 late in conjugation delayed chromosome breakage and decreased the overall amount of breakage observed. Loss of Dcl1 late in conjugation resulted in a complete loss of chromosome breakage. ChIP experiments also indicate that unlike sites of IES excision, chromosome breakage sites are not demarcated with the histone modification H3K9me3. These results indicate the differences between the two chromosome processing events but also suggest that there may be some overlap in the proteins required for these distinct pathways.

Carolyn Price (Committee Chair)
Andreassen Paul, PhD (Committee Member)
Rhett Kovall (Committee Member)
William Miller (Committee Member)
James Stringer (Committee Member)
167 p.

Recommended Citations

Citations

  • Heyse, S. R. (2011). Functional divergence between Tetrahymena telomere proteins: Potential role for POT1b in chromosome breakage and new telomere synthesis [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1299168952

    APA Style (7th edition)

  • Heyse, Serena. Functional divergence between Tetrahymena telomere proteins: Potential role for POT1b in chromosome breakage and new telomere synthesis. 2011. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1299168952.

    MLA Style (8th edition)

  • Heyse, Serena. "Functional divergence between Tetrahymena telomere proteins: Potential role for POT1b in chromosome breakage and new telomere synthesis." Doctoral dissertation, University of Cincinnati, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1299168952

    Chicago Manual of Style (17th edition)