Characterizing DNA Repair Pathways and Proteins

This summer, Colgate student researchers joined Assistant Professor of Biology Sarah McMillan to investigate a protein akin to a first responder. McMillan's lab focuses on single-stranded binding protein (SSB) found in Escherichia coli (E. coli) and its role in repairing damaged DNA.

McMillan's lab aims to further characterize the role of SSB as a first responder and the proteins that are part of the crisis response. "We are both identifying new SSB-interacting proteins and characterizing proteins we already know interact with SSB," McMillan explains.

When DNA damage is detected, SSB rapidly binds to any single-stranded DNA within a cell. Much like first responders working collaboratively with other resources, SSB recruits more than 20 other proteins to help repair the damage. Understanding DNA repair in bacteria is important because, when these processes go wrong, bacteria can develop mutations that allow them to build resistance to antibiotics.

This summer's research project centers on that coordinated effort to repair damaged DNA, with different proteins relying on each other to keep a cell's genetic code intact. Three students - Nathan Molloy '27, Katherine Cline '27, and Tolu Ladapo '28 - are working with McMillan to characterize DNA repair pathways and proteins involved in the process.

This spring, McMillan's research tutorial students identified what they believed to be a binding site between SSB and Exonuclease IX (ExoIX), an important DNA repair enzyme that removes damaged bases so new ones can be synthesized in their place. This gave a starting point for summer research students, each of whom has taken different approaches to understanding the SSB protein and its binding site on ExoIX.

Nathan Molloy '27: Breaking the Bond on Purpose

Molloy, a biology major and pre-medicine student, aimed to further characterize the relationship between SSB and ExoIX this summer. During his first two weeks as a research assistant, Molloy and Ladapo purified SSB and ExoIX and established binding assays between the two proteins. Then, Molloy strategically mutated ExoIX - changing small portions of ExoIX - to see how it impacts the strength of attraction between SSB and ExoIX. The ultimate goal of this research is to understand how disrupting the SSB-ExoIX interaction impacts bacteria's ability to repair DNA damage.

Research has improved Molloy's foundational laboratory skills, building confidence through repetition. "I'm definitely becoming more independent with a lot of the laboratory techniques that are common in the field," Molloy says.

Katherine Cline '27: Building the Map

Cline, a biology major and global public and environmental health minor, is approaching the same binding site with a different method. Cline uses AlphaFold, an AI model that predicts how proteins interact. After using AlphaFold, Cline uses an assay called yeast two-hybrid to determine whether the prediction was correct.

This was Cline's first summer as a research assistant. "It's very independent - Prof. McMillan lets you brainstorm and really do the behind-the-scenes work you wouldn't normally do in a typical lab," Cline says. She's now weighing a PhD among other options on her list of possible plans.

Tolu Ladapo '28: Putting Theory to the Test

Unlike Molloy and Cline, who are already probing known interactions, Ladapo, a molecular biology major, is researching a third binding protein called SbcC, which is involved in repairing severe types of DNA damage. It has never been shown to interact with SSB before, but results from a recent protein-protein interaction screen suggest that SbcC binds to SSB. Throughout the summer, Ladapo investigated whether the interaction between SSB and SbcC exists using various biochemical techniques.

During the last three weeks of research, Ladapo and other researchers helped McMillan prepare for her research tutorial students alongside their own experiments. "It takes a week for us to do it, and we're there all day," Ladapo says, explaining the time-intensive nature of this step.

What's Next

McMillan's research tutorial students - including Cline and Molloy - will continue this research in the fall. Ultimately this work will help reveal how DNA repair efforts are coordinated in bacteria and the important mechanisms for preventing bacteria from acquiring resistance to antibiotics.