Study of mechanism of genetic death in Escherichia coli in response to A) dTTP starvation and B) combination of temperature sensitive defects in replication initiation and double strand break repair

Rao, Pritha

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Description

Title

Study of mechanism of genetic death in Escherichia coli in response to A) dTTP starvation and B) combination of temperature sensitive defects in replication initiation and double strand break repair

Author(s)

Rao, Pritha

Issue Date

2021-04-07

Director of Research (if dissertation) or Advisor (if thesis)

Kuzminov, Andrei

Doctoral Committee Chair(s)

Kuzminov, Andrei

Committee Member(s)

• Imlay, James A.
• Blanke, Steven R.
• Whitaker, Rachel J.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Thymineless Death
• Genetic Death
• E. coli
• DNA Replication
• DNA Repair

Abstract

At the cellular level life can be described as replication of metabolism. Cells first grow by continuous synthesis of RNA, protein and lipids. Once they reach a certain dimension, they duplicate their genomes, which is followed by the division of the mother cell into two daughter cells, capable of repeating this growth – duplication – division cycle again. Because of its dual nature, this cycle can be stopped by either inhibiting the period of continuous cellular growth (i.e. preventing the synthesis of RNA, proteins, lipids), which can be classified as metabolic death of the cell, or by blocking the process of DNA replication and segregation (without affecting protein/RNA/lipid metabolism) – which we classify as genetic death of the cell. Irreparable DNA lesions, therefore, inflict genetic death on the cells, and we are interested in identifying such DNA lesions or conditions that lead to them, providing future effective strategies to get rid of cancerous or infecting cells. In this thesis, I present our investigation of two distinct examples of genetic death in E. coli. The main story is about death in response to dTTP starvation known as Thymineless Death (TLD). This phenomenon has been subject to decades of investigation since its discovery in 1954, and I present our recent efforts to further understand the consequences of dTTP starvation in prokaryotic cells. The other example of genetic death that I studied is the synthetic lethality of the combination of two partial defects in E. coli – dnaA in replication initiation and recBC, in double-strand-break repair. This lethality was discovered by Sharik Kahn, a Post-Doctoral researcher in this lab, and I continued to investigate this mysterious synthetic lethal combination. The first chapter presents a review of the phenomenon of TLD, the history of its investigation and a summary of recent progress in the elucidation of its complicated mechanism. The second chapter presents my work on TLD which clarifies the role of non-canonical nucleotide incorporation-excision cycles on DNA stability and viability of E. coli cells. Additionally, we explore some possible sources of endogenous dT E. coli cells utilize in the later stages of dTTP starvation. We also report detection and accumulation of persistent single-strand gaps that happen concurrently with viability loss due to dTTP starvation. In the third chapter, I report novel mutants that experience hyper-TLD and cell lysis when E. coli cannot utilize an overlooked internal source of dTTP. We show that E. coli relies on this internal reserve of dT to maintain its viability early during dTTP starvation. We also confirm the (discovered earlier) replication-independence of TLD and propose a possible mechanism of replication-independent chromosome fragmentation observed in hyper-TLD mutants. The fourth chapter is a compilation of TEM images of E. coli cells undergoing TLD. As a comparison, we have also imaged E. coli cells treated with nalidixic acid, which kills the cells by chromosome damage unrelated to TLD. The fifth chapter presents my investigation on the synthetic lethal combination dnaA recBC and another known colethal combination dnaN recBC. We find that dnaA mutants require DNA degradation activity of RecBCD for viability, while dnaN relies more on recombinational repair for its viability. We show that replication is stalled in the dnaA recBC mutant, while it is inhibited but continues in the dnaN recBC mutant. We are yet to identify the DNA lesion responsible for fork stalling in the dnaA recBC mutant. In the final chapter, I summarize my overall findings and propose specific questions to pursue in the future, to further explore the phenomena of TLD and dnaA recBC colethality.

Graduation Semester

2021-05

Type of Resource

Thesis

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Copyright 2021, Pritha Rao

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