Uncovering pathways necessary for the survival of RNase H-deficient Escherichia coli

Ribonucleotides (rNs) frequently contaminate DNA, forming RNA-DNA hybrids, (RDHs) which cause genomic instability if not removed. Consequently, all organisms are equipped with RNase H enzymes to remove RDHs. Escherichia coli lacking both the RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ΔrnhA ΔrnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that interfere with chromosome replication and segregation. As a result, the ΔrnhAB double mutant has severe phenotypes, including growth inhibition, replication stress, sensitivity to ultraviolet (UV) radiation, SOS induction, increased chromosomal fragmentation, and defects in the nucleoid organization. In this work, I found that RNase HI deficiency also increases the levels of DNA supercoiling. Despite these serious chromosomal complications, the ΔrnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, I systematically searched for mutants synthetically lethal (colethal) with the rnhAB defect, using an unbiased color screen and a candidate gene approach. I identified both novel and previously reported colethal and coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. Anaerobic lethality of the ΔrnhAB double mutant is an intriguing phenotype because RNase H enzymes have no known role in anaerobic metabolism. I isolated spontaneous suppressors to identify the genes/pathways involved. Two unexpected primary suppressors are mutations in genes rnlA (the toxin in the RnlAB toxin-antitoxin system) and hscA (a chaperone essential for Fe-S cluster assembly); one of the expected phenotypes of the hscA inactivation is a decreased expression of the rnlA gene.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Das, Sneha

Contributors dc:contributor

• Kuzminov, Andrei
• Brooke, Christopher B.
• Imlay, James A.
• Cronan, John E.

• Dna Supercoiling
• Rnase H
• Avoidance And Repair Pathways
• Genetic Screen
• Nucleoid Organization
• Rnhab
• Synthetic Lethal
• Trna Modification
• Anaerobic Lethality.

Statement dc:rights

• Copyright 2024 Sneha Das

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/124625

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F124625