First Advisor

Justin Courcelle

Date of Award

Spring 6-2025

Document Type

Thesis

Degree Name

Bachelor of Science (B.S.) in Biology and University Honors

Department

Biology

Language

English

Subjects

DNA replication, Transcription-coupled repair, Replication-coupled repair, Nucleotide excision repair, Bacterial two-hybrid system, HolC

Abstract

Cells rely on nucleotide excision repair (NER) to remove bulky DNA lesions, such as those caused by UV light. In Escherichia coli, NER involves the UvrABC complex, and includes two main pathways, global genomic repair and transcription-coupled repair (TCR). While TCR, which preferentially removes lesions that block RNA polymerase on the transcribed strand of active genes, is well studied, less is known about whether a similar coupling between repair proteins and replisomes blocked by damaged DNA exists. Such a process has been suggested by a high throughput interactome study, using the replisome subunit HolC as bait and the damage recognition repair protein UvrA as prey, but this interaction has not been verified. Here, I use the Bacterial Adenylate Cyclase-Based Two-Hybrid system to look for a direct protein-protein interaction between UvrA and HolC. Genes encoding HolC, UvrA, and UvrB were cloned into plasmid vectors to create fusion proteins that were tagged with either the T18 or T25 subunits of adenylate cyclase at their carboxyl terminus. Interactions between T18 and T25-fused proteins were then screened by observing the color of colonies produced on indicator agar plates. Blue colonies produced by co-transformations indicate a positive protein-protein interaction while white colonies indicate no protein-protein interaction. I observed blue colonies in all hybrid protein pairings involving UvrA-T18 as bait, even when the partner protein used was the T25 subunit alone, suggesting a high level of background expression from the reporter gene that was independent of a true protein-protein interaction. I discuss some experimental modifications that can be made to address the non-specificity of this assay.

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