Degree Type

Dissertation

Date of Award

2003

Degree Name

Doctor of Philosophy

Department

Zoology and Genetics

Major

Molecular, Cellular, and Developmental Biology

First Advisor

Daniel Voytas

Abstract

The condensed structure of heterochromatin differs significantly from euchromatin and DNA bound in heterochromatin is generally not transcriptionally active. Transcriptional silencing allows the control of gene expression and affords a novel mechanism of defense against genomic parasites. In addition, the physical structure of heterochromatin plays a direct role by contributing to centromere function in many eukaryotes. Mechanisms by which heterochromatin is established and maintained are only partly understood at this time. By studying the interaction of Ty5, a retrotransposon that integrates preferentially into heterochromatin-bound DNA, some of the basic biology of heterochromatin and the proteins that compose it were elucidated. The interaction between a portion of the Ty5 integrase and Sir4p, a component of yeast heterochromatin, was used to nucleate a heterochromatin complex. This complex was found to antagonize centromere-mediated plasmid segregation in a fashion similar to that reported between centromeres and other heterochromatin complexes. This antagonism was further found to be the result of double strand breaks in the plasmid DNA. The interaction between the Ty5 integrase and Sir4p was more fully mapped and four Sir4p amino acid residues were found to be required for the interaction. When mutations in these residues were introduced into the full-length SIR4 allele, the function of SIR4 in heterochromatic silencing was impaired. This is likely due to a decrease in Sir4p protein levels. The interaction between Sir4p and a short fragment of integrase required for targeting (LDSSPP), referred to as the targeting domain (TD) was also examined. This interaction was found to partially depend on other components of heterochromatin. Two-hybrid analysis of the integrase interacting portion of Sir4p identified three novel interacting proteins, Sum1p, Nma2p and Chd1p. Mutations in Sir4p required for the interaction between Sir4p and integrase were found to be required for the interaction between Sir4p and several other Sir4p interacting proteins. Expression of the targeting domain TD was also found to cause anti-silencing by lowering Sir4p levels. Genetic analysis identified the RING finger protein Ris1p as a required factor in the Sir4p turnover. These findings suggest that the previously identified role for Ris1p in gaining access to heterochromatin is likely mediated by Sir4p turnover.

DOI

https://doi.org/10.31274/rtd-180813-4113

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Peter Fuerst

Language

en

Proquest ID

AAI3089110

File Format

application/pdf

File Size

127 pages

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