Doctoral Dissertations

Date of Award

12-2004

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Chris Dealwis

Committee Members

Robert Hettich, Jeffrey Becker, Engin Serpersu, Elizabeth Howell

Abstract

Sml1 is a small protein in Saccharomyces cerevisiae that inhibits the activity of ribonucleotide reductase (RNR) through its interactions with the large subunit of RNR. RNR catalyzes the reduction of nucleotide diphosphates (NDPs) to deoxynucleotide diphosphates (dNDPs) that is the rate-limiting step of de novo deoxynucleotide triphosphate (dNTP) synthesis. The cellular level of Sml1 is regulated by DNA damage and replication block response through its phosphorylation by the Dun1 kinase. The goal of this dissertation research is to elucidate structure-function and regulation of Sml1. First, biochemical characterization of recombinant Sml1 was conducted using mass spectrometry and gel filtration chromatography (Chapter 3 and 4). The data shows that a disulfide bond and non-covalent interactions mediate Sml1 oligomerization. Furthermore, alkali metal adducts (Na+/K+) that bind strongly with Sml1 were found. Second, the phosphorylation of Sml1 by the Dun1 kinase was studied by a combination of mass spectrometry, site directed mutagenesis, and P32 incorporation (Chapter 5). Three phosphorylation sites of Sml1 (Ser56, Ser58 and Ser60) were identified. The data also reveals that the Dun1 kinase requires an acidic residue at the +3 position and there is cooperativity between the phosphorylation sites. Third, the relationship between Sml1 phosphorylation and the Sml1-Rnr1 interactions were investigated based on P32 incorporation, fluorescence spectroscopy, and a RNR activity assay (Chapter 6). We demonstrated that the Sml1-Rnr1 interactions reduced the phosphorylation levels of Sml1 by making the phosphorylation sites less accessible. Our data also suggest that phosphorylation of Sml1 weakens the ability of Sml1 to inhibit RNR. Taken together, this work has provided in-depth insights of Sml1’s structure-function and regulation.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS