The role of error-prone polymerases in mutagenesis and evolution

Project Summary
Mutations are the ultimate source of genetic variation in all organisms and, coupled with natural selection,
shape the natural world and are responsible for the biodiversity we enjoy today. However, mutations are crucial
for human health and disease. It is mutations that allow bacteria to become resistant to antibiotics and cancer
cells to chemotherapy. However, what cellular processes promote spontaneous mutagenesis, and therefore
drive evolution, as well as the role of the environment in this process, remains poorly understood. We have
shown that error-prone DNA polymerases promote mutations and the evolution of antibiotic resistance in
bacteria. Importantly, this is dependent on a DNA repair pathway called transcription-coupled repair, that
depends on transcription, but it is independent of replication. This is in contrast with the most well-known
mechanisms of mutagenesis, which depend on replication. Because the chemical makeup of the environment
in which different bacteria are found can dramatically affect the sources of DNA damaged that they are
exposed to, I hypothesize that it will have a strong influence on this pro-mutagenic process. The goal of this
proposal is to describe how bacterial error-prone polymerases promote transcription associated mutagenesis
and the evolution of antibiotic resistance, as well as how the environment in which bacteria live affects this
process. For the K99-phase of the proposal, I will characterize the molecular mechanism by which these
polymerases promote transcription associated mutagenesis (Aim1), and which types of mutations these
polymerases make in model bacteria (Aim2). At the same time, I will learn the necessary skills to study this
process in different medically relevant bacterial species. With these new skills, during the R00 phase of the
proposal I will determine which types of transcription associated mutations most commonly arise in highly
divergent bacteria that are found in different environments (Aim2). In addition, I will determine the evolutionary
history of these pro-mutagenic polymerases to find out how their environment has shaped the way they have
evolved (Aim3). In addition, I will establish the role of error-prone polymerases in driving bacterial evolution,
antibiotic resistance, and pathogenicity (Aim3). This research as well as my career development plan will take
part at Vanderbilt University, in the laboratory of Dr. Houra Merrikh. Dr. Merrikh has a track record of doing
groundbreaking work on the mechanisms of mutagenesis and the evolution of antibiotic resistance. During my
time in her lab, I will benefit from her expertise and scientific knowledge. The work pertaining to Aim3 will be
done with the assistance of my collaborator Dr. Antonis Rokas of Vanderbilt University, which is an expert in
the study of evolution. The novel skills I will learn during my time at Vanderbilt University will help me establish
an independent research program. Overall, this K99/R00 proposal will allow me to successfully transition from
a mentored scientist into a successful independent researcher, while providing key insights on how the
environment affects bacterial evolution and antibiotic resistance.

Grant Number: 1K99ES037493-01
NIH Institute/Center: NIH
Principal Investigator: Juan Carvajal Garcia