Timothy O'Connor, Ph.D.

DNA repair is a basic necessary function in all cells, and the mechanisms for repair or damage avoidance are evolutionarily conserved. Cells are constantly subjected to DNA damage from exogenous environmental sources, and also from endogenous oxidative metabolism. Our laboratory is interested in DNA repair mechanisms, the biological consequences of repair failure, and how DNA repair mechanisms can be used to control the epigenome of cells. A complete understanding of DNA repair pathways and the activities of DNA repair proteins can lead to the identification of cellular defects linked to cancer etiology or to targets for tumor therapy.

 

Another more recent area of interest for our laboratory is DNA repair in stem cells. Although stem cells hold great promise in human disease treatment, the DNA repair capabilities must be robust and the genetic stability of cells fully characterized before their use in any regenerative therapy.

 

To address these areas, we employ both in vitro and in vivo models to examine:

DNA in cells is constantly exposed to damage from both endogenous and exogenous sources. To remove damage and maintain genomic stability, cells have evolved DNA repair systems. The protein levels in these pathways are finely tuned, and DNA damage may induce production of DNA repair proteins. We study DNA damage and repair from several aspects. Our work involves the study of adducts, the repair enzymes involved in adduct removal, how a repair system functions to remove an adduct, how repair systems interact, and finally the response of cells to DNA damage. One system that we focus on is the base excision repair (BER) pathway replacing mismatched or modified bases in DNA. BER is one of the most important systems in the elimination of endogenous DNA damage. The goal of our research is to understand how DNA repair proteins function to eliminate deleterious adducts from DNA and maintain genomic stability.

 

Our research is divided into several areas:

We have cloned and overproduced numerous DNA repair proteins, and our work in this area continues. We have used the homogeneous proteins to study their biochemical and enzymatic properties. DNA repair proteins are often associated in complexes to facilitate repair. We have recently identified an interaction between two DNA repair proteins involved in the initial steps of both the base and nucleotide excision repair pathways. This interaction could prove critical in directing repair along both pathways. We are currently developing other methods to study these protein-protein interactions.

In response to DNA damage, DNA repair capacity can increase, decrease, or remain unchanged. We are now investigating the response of DNA repair genes to DNA damage at the mRNA, protein, and activity levels. Alteration of the levels of DNA repair proteins can result in a change in the efficiency of a given DNA repair system to remove adducts. This work will serve as the basis for predicting the outcome of different chemo- and radio-therapeutic treatments.

In addition to the study of individual DNA repair enzymes, we are interested in how these enzymes function in cells to excise DNA damage. We are using genomic sequencing techniques, such as ligation-mediated polymerase chain reaction (LMPCR), to follow DNA repair in vivo. We have shown that the repair of methylated bases via BER at nucleotide resolution in normal human cells is heterogeneous and have identified sites of DNA repair footprints. Damage and repair at nucleotide resolution of bases damaged by oxidation and chemotherapeutic agents have also been studied. We are adapting this technique to study the effects of gene therapy agents on the function of DNA repair in human cells.