A Biochemical Analysis of Transcription-coupled Repair Proteins in Yeast and their Role in Transcription Elongation and mrna degradation

Open Access
Author:
Wing, Anna Kait
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Joseph C. Reese, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • biochemistry
  • transcription
  • transcription-coupled repair
  • DNA repair
  • mRNA degradation
  • Ccr4-Not complex
  • DNA damage
Abstract:
Transcription-coupled repair (TCR) is an essential part of the cell’s DNA repair machinery. It is the most effective way to fix damage caused by UV radiation and oxidative stress before dangerous or cancerous mutations can form. This mechanism is associated in S. cerevisiae with the Ccr4-Not complex, a complex of nine proteins that are highly conserved in eukaryotes from yeast to humans. In addition to activities related to the cell stress response, the Ccr4-Not complex plays a role in key pathways of transcription elongation and mRNA degradation as demonstrated by elongation and degradation defects present in Ccr4-Not mutants. However, the precise proteins that link these two processes are currently unclear. In this project, several TCR-associated proteins were analyzed to determine their role in transcription elongation and mRNA degradation through the quantification of gene-length dependent elongation rates and mRNA degradation rates in mutant strains of yeast. While none of the proteins analyzed had an effect on transcription elongation, two of the five proteins studied were shown to significantly impact mRNA degradation rates within the cell—Not4, a subunit of the Ccr4-Not complex, and Def1. The other three TCR-associated proteins analyzed, Ubp3, Bre5, and Rad26, did not demonstrate an effect and therefore are not implicated in the mRNA degradation pathways. Further study of the Not4 protein and its domains will be necessary to determine which protein domains are essential for efficient RNA turnover and to better understand the role of Def1 in linking transcription-coupled repair to mRNA degradation.