Type
Text
Type
Dissertation
Advisor
Nancy Hollingsworth | Stillman, Bruce | Bruce Futcher | Rolf Sternglanz | Rodney Rothstein.
Date
2010-08-01
Keywords
Biology, Genetics -- Biology, Molecular -- Biology, Microbiology | budding yeast, DNA damage response, DNA replication, heterochromatin, nucleotide metabolism, telomeric silencing
Department
Department of Genetics
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/72655
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
In budding yeast, telomere position effect variegation (TPEV) was discovered when prototrophic markers were placed near chromosome ends and was interpreted to reflect a reversible form of heterochromatin. Selection for or against these markers demonstrated roles for several proteins in telomeric heterochromatin formation; these include the SIR protein complex, chromatin assembly factors (CAF-1, Asf1), PCNA as a DNA replication factor as well as DNA damage checkpoint proteins. PCNA (POL30) in particular as a component of the core DNA replication machinery has been shown to link DNA replication to the inheritance of nucleosomes and was, by extension, proposed to help maintain silenced chromatin. In analyzing the phenotype of the silencing defective pol30-8 mutant using various TPEV reporter strains I found that this mutant exhibits only a very subtle telomeric silencing defect in comparison to a sir3&Delta mutant. Furthermore, employing the common URA3 reporter at the telomere of chromosome VIIL that can be counter-selected with 5-FOA in a genetic screen, I identified high-copy suppressors of the pol30-8-dependent silencing defect. Interestingly, one of the suppressors, CDC21, the thymidylate synthase gene, also counteracted the telomeric silencing defect of a strain deleted for DOT1, encoding the only histone H3K79 methyltransferase in S. cerevisiae. Gene expression analysis of pol30-8 mutant and dot1 mutant strains surprisingly revealed that dot1&Delta deletion results in repression of telomeric gene expression without an effect on Sir2/4 occupancy. On the other hand, the pol30-8 mutation was linked to a general up-regulation of normally poorly expressed genes. Notably, the effect of pol30-8 correlated with decreased histone levels. Among the affected genes were the ribonucleotide reductase (RNR) genes whose expression could be further induced by treatment with 5-FOA. Importantly, inhibition of RNR activity as well as mutations in the RAD53 DNA damage response pathway rescued the sensitivity of pol30-8 URA3-VIIL cells to 5-FOA. I speculate that in the context of low URA3 expression such as in a URA3-VIIL strain a misbalance between Ura3, RNR and Cdc21 activity is responsible for a higher conversion rate of 5-FOA into its toxic metabolites which accounts for the 5-FOA sensitivity seen in pol30-8 and dot1&Delta URA3-VIIL mutants. In conclusion, while I found that Pol30 facilitates normal histone distribution with consequences for global gene expression, neither pol30-8 nor dot1&Delta mutants are defective in telomeric heterochromatin formation.
Recommended Citation
Rossmann, Marlies Petra, "A common telomeric gene silencing assay is affected by nucleotide metabolism and the DNA damage response: implications for the role of PCNA in heterochromatin formation." (2010). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 1858.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/1858