A Biochemical Analysis of the Senataxin Protein 公开

Abstract

Abstract
A Biochemical Analysis of the Senataxin Protein
By Brian F. Gilmore

The yeast protein Sen1 is essential for cell viability. Although its essential function is unknown, homology analysis has established a substantial sequence similarity between the C-terminal region of Sen1 and Superfamily I helicases (Steinmetz et al. 2006). Additionally, this region of helicase homology has been established as the same region required for cell viability. However, helicase activity has only been demonstrated in the Sen1 protein of Schizosaccharomyces pombe (Kim, et al., 1999).

The human ortholog of Sen1, senataxin, is poorly understood. However, positional cloning studies have established that mutations in the senataxin-encoding SETX gene are linked to the neurodegenerative disorders AOA2 and ALS4. The positions of these mutations typically result in truncated proteins lacking the helicase homology domain or mutations within the helicase motifs, although other mutations have been identified (Bassuk et al. 2007). However, without additional knowledge of the biochemical function of senataxin, the functional effects of the mutations remain unknown. Thus, the effort to determine the function and significance of the Senataxin protein in humans is framed by the need to understand the functional significance of these disease-causing mutations.

Herein, we analyze the functional biochemistry of senataxin and provide some evidence that the protein exhibits helicase activity in vitro and that this activity is lost in disease-linked mutants. Additionally, our results indicate that senataxin may function as a nucleic acid-stimulated ATPase under specific conditions. However, these findings are thus far inconclusive and additional studies are required to confirm the in vitro function of senataxin and how this relates to its essential in vivo role.

Table of Contents

Introduction ............................................. 1

1. Helicases ............................................. 1

2. Sen1 ................................................... 2

3. Senataxin ............................................ 4

4. Background .......................................... 5

5. Specific Aims ........................................ 7

Methods and Materials ............................... 8

1. E. coli Expression Constructs ................... 8

2. Protein Production and Purification ........... 11

3. Helicase Assay ..................................... 12

4. ATPase Assay ...................................... 14

Results ................................................... 15

Conclusions ............................................ 26

References ............................................. 31

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Chemistry, Biochemistry Biology, General
关键词	Biochemistry Senataxin Helicase
Committee Chair / Thesis Advisor	Reines, Daniel, Emory University
Committee Members	Crouse, Gray, Emory University Yedvobnick, Barry, Emory University

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