This research program stems from our past fundamental studies of DNA charge transport (DNA CT) chemistry. This chemistry facilitates redox chemistry at a distance through the DNA duplex and sensitively depends upon the integrity of the intervening base pair stack. Our goal now is to apply this chemistry for the sensitive sensing of DNA lesions and DNA processing events electrically and, importantly, to determine general characteristics of how DNA CT chemistry is utilized within the cell for long range chemical signaling. In particular, these studies may establish a new framework for considering the role of [4Fe4S] clusters in critically important enzymes that process DNA. With respect to sensing, we will continue in the development and application of our multiplexed platform in developing new electrochemical strategies for the detection of DNA-binding proteins and nucleic acid markers associated with cancer. In general, our multiplexed DNA electrochemical platform offers a completely new approach to sensitive, well-controlled detection of DNA-binding events. Our overall goal is the development of highly sensitive sensors based on DNA CT chemistry that can be used to monitor biomolecules in a single cell. Significantly, our goal also is to understand how Nature may use DNA CT; we will continue our studies to characterize the redox chemistry of critically important DNA- processing enzymes involved in replication and DNA repair to determine how this chemistry may be utilized for long range signaling across the genome. We will use DNA electrochemistry anaerobically using multiplexed DNA chips to characterize DNA-processing proteins containing redox-active [4Fe4S] cofactors. Overall, experiments we have carried out thus far provide support that DNA CT signaling is a means for diverse proteins with different roles in DNA processing to coordinate their activities as a team. DNA CT may be used for polymerase hand-offs and to coordinate a rapid response to oxidative stress. We will build upon this foundation by exploring DNA CT signaling among protein teams involved in DNA transcription and replication as well as repair, using AFM assays, genetics, and spectroscopy on [4Fe4S] proteins and mutants.
We aim to use our expertise in the electrochemical and biochemical characterization of DNA CT proficiency to identify new CT-active protein players and illuminate the DNA CT signaling networks in which they participate. This work will contribute both to our fundamental understanding of protein/DNA signaling and the important consideration of DNA CT deficiencies associated with disease.
This proposal describes the elucidation of long range DNA signaling by DNA-processing proteins and its application in developing new DNA sensors for cancer markers. DNA-processing proteins coordinate DNA replication and its repair when the DNA is damaged. Mutations in these processes are associated with cancer and correcting these diseases requires understanding this chemistry. !
|Holt, Marilyn E; Salay, Lauren E; O'Brien, Elizabeth et al. (2018) Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs. PLoS One 13:e0209345|
|O'Brien, Elizabeth; Salay, Lauren E; Epum, Esther A et al. (2018) Yeast require redox switching in DNA primase. Proc Natl Acad Sci U S A 115:13186-13191|
|O'Brien, Elizabeth; Holt, Marilyn E; Salay, Lauren E et al. (2018) Substrate Binding Regulates Redox Signaling in Human DNA Primase. J Am Chem Soc :|
|Ekanger, Levi A; Oyala, Paul H; Moradian, Annie et al. (2018) Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation. J Am Chem Soc 140:11800-11810|
|McDonnell, Kevin J; Chemler, Joseph A; Bartels, Phillip L et al. (2018) A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster. Nat Chem 10:873-880|