Date of Award

5-1-2008

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Cell Biology and Genetics

Department

Graduate School of Biomedical Sciences

First Advisor

Wolfram Siede

Second Advisor

Rafael Alvarez

Third Advisor

Rustin Reeves

Abstract

Pabla, Ritu., Tolerating DNA damage: Translesion polymerase eta (η) and its regulation in Saccharomyces cerevisiae. Doctor of Philosophy (Cell Biology and Genetics), May 2008, 137 pp., 21 illustrations, bibliography, 151 titles. RAD30 gene encoded DNA polymerase eta (Polη) is the only eukaryotic polymerase that can bypass UV-induced thymine-thymine (T-T) dimers in a predominantly error-free manner. The unique ability of reading bulky and geometrically distorted bases in the template makes the polymerase low-fidelity and error-prone for an undamaged template. The purpose of this study is to delineate the mechanism(s) by which activity of Polη is regulated. The increase in RAD30 transcript after UV damage is not reflected at the protein levels. Instead, Polη is monoubiquitinated constitutively. This posttranslational modification is upregulated in G1 phase and downregulated on entry into S phase of the cell-cycle. This downregulation is further accelerated in response to UV induced DNA damage. A missense mutation (L577Q) of the ubiquitin binding domain (UBZ) results in reduced degree of ubiquitination of the mutant protein outside of G1 and a complete failure to stably interact with ubiquitinated substrates. This mutation renders the strain more UV sensitive and mutagenic, a phenotype resembling a complete RAD30 deletion. In other words, UBZ motif and its interaction with ubiquitinated PCNA is critical for Polη function in vivo. In addition to nucleus, the polymerase localizes in mitochondria suggesting its role in damage tolerance in mitochondria. No drastic changes in the localization of polymerase are observed during cell-cycle progression and after UV damage.

Comments

Pabla, Ritu., Tolerating DNA damage: Translesion polymerase eta (η) and its regulation in Saccharomyces cerevisiae. Doctor of Philosophy (Cell Biology and Genetics), May 2008, 137 pp., 21 illustrations, bibliography, 151 titles. W 4 P112T 2008

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