Dysregulated DNA replication forks as targets for formaldehyde mutagenesis

Medical University Of South Carolina, Charleston SC

Investigators

Abstract

Project Summary Formaldehyde is a class I carcinogen and is present in the environment and generated endogenously by cells. Formaldehyde has been shown to form DNA-base adducts and DNA-protein crosslinks, both of which contribute to formaldehyde-induced genomic toxicity. My preliminary data using an inducible single-stranded DNA (ssDNA) system in yeast has shown that formaldehyde preferentially mutagenizes ssDNA and that this mutagenesis is dependent on translesion synthesis and DNA-protein crosslink repair. Currently, it is unknown which sources of endogenous ssDNA act as targets for formaldehyde-induced mutagenesis. Without this knowledge, it is impossible to know which individuals are at elevated risk for formaldehyde-induced carcinogenesis. One of the major sources of ssDNA is at replication forks. While ssDNA production at replication forks is typically tightly regulated, several human disorders cause fork dysregulation and ssDNA accumulation. Therefore, my central hypothesis is that ssDNA generated at dysregulated replication forks is a preferential substrate for formaldehyde- induced mutagenesis. Aim 1 will employ a sensitive mutational reporter in yeast to investigate dysregulated replication forks as substrates for formaldehyde-induced mutagenesis. This will be done via replicative polymerase knockdown and manipulation of the replication fork protection complex. In aim 2, I will investigate replication-associated ssDNA as a preferred formaldehyde substrate in human HepG2-aldh2-/-. Replication stress will be induced with hydroxyurea and cells will be treated with formaldehyde to determine a formaldehyde- induced mutational signature in human cells. Additionally, cells will be treated with the translesion synthesis inhibitor JH-RE-06 (REV1) or the DNA-protein crosslink repair inhibitor 1,10-Phenanthroline (SPRTN) to determine the roles of these pathways on formaldehyde mutagenesis in human cells. Using this combination of yeast reporter systems and human cells, I will investigate replication forks as preferential targets for formaldehyde-induced mutagenesis. The findings of this work will be significant because it will identify individuals who are “at-risk” for formaldehyde-induced carcinogenesis and establish a formaldehyde-specific mutational signature in human cells that can be used to track long-term environmental exposures to formaldehyde. This research will be performed at the Medical University of South Carolina under the mentorship of Dr. Natalie Saini and Dr. David Long. The Department of Biochemistry and Molecular Biology and the Hollings Cancer Center both have well established and effective programs for student training. Being the only Ph.D. student in the Saini lab, I have gotten an extremely personalized and hands on training experience, and Dr. Long contributes significant mentorship experience to my training plan. This proposal outlines the experimental and professional development goals for my training as I prepare for a career as an independent principal investigator at a research-focused institution.