The role of ClpXP-mediated proteolysis in resculpting the proteome after DNA damage

When faced with environmental assaults, E. coli can take extreme measures to survive. For example, starving bacteria consume their own proteins, and bacteria with severe DNA damage introduce mutations into their genomes. These survival tactics require restructuring of the bacterial proteomic landscape. To reshape the proteome, bacteria alter both protein synthesis and protein degradation. For important regulatory proteins and proteins potentially harmful to the cell under non-stress conditions, these changes must be environmentally responsive and specific. This thesis explores the role of the ClpXP protease in the response to DNA damage. First, we determine how DNA damage affects substrate selection by ClpXP. These experiments combine quantitative proteomics and use of an inactive variant of ClpP to "trap" cellular ClpXP substrates and compare their relative levels with and without DNA damage. Analysis of trapped substrates reveals that cellular stress can result in dramatic changes in protease substrate selection. Next, we explore a specific mechanism that allows coupling of an environmental signal to a change in proteolysis. When the cell senses DNA damage, it triggers autocleavage of the LexA repressor. Autocleavage creates new signals for ClpXP recognition, ensuring the timely degradation of the LexA cleavage products. The mechanism of LexA recognition became a model for cleavage-dependent recognition of other substrates. Finally, we determined the mechanism of ClpX recognition of a known, damage-inducible substrate, UmuD'. We find that UmuD directs UmuD' degradation in an SspB-like manner. These experiments show how, with the right sequence motif, an interacting partner can become a ClpXP delivery factor.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Neher, Saskia B. (Saskia Byerly)

Advisor dc:contributor.advisor

• Tania A. Baker.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/31188

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/31188