Uncovering vital molecular contacts within the replisome and characterizing fail-safe mechanisms ensuring genomic stability in E. coli.

To study replisome coupling interactions and gain a better understanding of enzymatic communication, we modeled interfacial residues between α-Pol III and τ-CLC. A critical C-terminal linker in the τ-subunit tethers α-Pol III to the DnaB helicase for efficient DNA replication in E. coli. Therefore, disrupting interactions between τ-CLC and α-Pol III also disrupts coordination with the DnaB helicase. To probe this phenomenon, we initiated our study through in vitro investigations by using site-directed mutagenesis techniques to alter the following residues in α: L1097S, Y1119A, L1097/8S, and L1128S. We then purified these α-Pol III mutants: 1) to verify site-specific interactions between τ-CLC and α-Pol III by probing target residues through nickel pull-down assays, and 2) to quantify differences in unwinding and polymerization activities amongst wild-type and mutant enzymes by perturbing DNA unwinding and synthesis coupling with an in vitro assembled replisome. Once we confirmed that each of these mutations disrupted physical interactions with τ and severely compromised DNA synthesis abilities in whole replisome rolling circle experiments, we created analogous genomic mutants of α-Pol III in vivo using CRISPR-Cas9. Our in vivo investigations uncovered severe cellular and genomic fitness deficits. Decoupling between enzymes led to severe cellular stress phenotypes, characterized by reduced growth, lower fitness, heightened stress, SOS induction, accumulation of ssDNA gaps, and increased cell death. Without effective coupling, labile ssDNA accumulates and is left exposed. However, E. coli has numerous mechanisms to circumvent decoupling, including multiple replication restart pathways to rescue compromised cells. The Rep helicase can rescue abandoned replication forks by remodeling the fork to reload DnaB directly. Deletion of Rep (Δrep) reduces ssDNA gaps in untreated cells; however, in the presence of various genotoxins, the absence of Rep results in an increase in ssDNA gaps due to DNA damage-linked replisome decoupling. By characterizing decoupling consequences within the E. coli replisome, we have gained a deeper, more comprehensive understanding of these dynamics and essential interactions that contribute to the overall health and survivability of the cell. Taken together, we’ve demonstrated that replisome communication between these enzymes is crucial for overall health and the maintenance of genomic information and integrity.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Grantor

Baylor University.

Year dc:date.issued

2025

Author dc:creator

• Butterworth, Lauren Julia, 1995-

Advisor dc:contributor.advisor

• Trakselis, Michael A.

• E. coli.
• Enzyme coupling interactions.
• Genomic integrity.
• Polymerase III holoenzyme.
• Replication restart.

Statement dc:rights

• Baylor University works 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. Contact libraryquestions@baylor.edu for inquiries about permission.

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2104/13964

OAI identifier oai:identifier

oai:baylor-ir.tdl.org:2104/13964