Dr. Eric Josephs (Joint School of Nanoscience and Nanoengineering) received new funding from the NIH National Institute of General Medical Sciences for the project “Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells.”
All organisms strive to maintain genomic fidelity in the face of agents that can damage their genetic material and the possibility that errors that can occur whenever their DNA is replicated. The ultimate goals of this research are to understand (i) how the mechanisms and higher-order coordination of DNA repair processes are governed by molecular, genetic, and epigenetic factors in vivo; (ii) how these factors affect diverse repair processes in different contexts to affect human health; and (iii) how clinically-important modulators of DNA repair activities and of repair-related toxicity can be leveraged as novel therapeutics.
Josephs has focused primarily on DNA mismatch repair (MMR) pathways, the pathways responsible for correcting errors that occur during DNA replication. As a primary mechanism of DNA damage repair in nearly all organisms, MMR plays a central role in many diverse processes that affect human health, from the emergence of drug resistance in infectious pathogens and cancers to the onset and treatment of somatic genetic diseases.
The researchers developed a novel assay to deconstruct the mechanisms of MMR in vivo that uses chemically-modified oligonucleotide probes to insert targeted DNA ‘mismatches’ directly into the genome of living cells. This assay, which they call by the acronym ‘SPORE,’ can therefore be used to directly interrogate replication-coupled repair processes like MMR quantitatively in a strand-, orientation-, and lesion-specific manner—something otherwise nearly impossible to achieve. Using the SPORE assay as a uniquely powerful baseline of approach, in combination with next-generation biotechnologies like CRISPR and innovative experimental design, the researchers will seek to answer the following questions:
- How do different molecular, genetic, and epigenetic factors affect the higher-order architecture (components and interactions), coordination, dynamics of canonical and non-canonical MMR mechanisms?
- How do these factors affect repair-associated toxicities? Are different molecular lesions recognized by MMR repaired according to different mechanisms with different toxicities?
- Do the unique repair mechanisms in pathogenic organisms represent a novel source of antimicrobial targets?
- How do viral factors and environmental mutagens modulate MMR and MMR-related toxicities and by what mechanism?
- What is their role in hypermutation and emergence of drug resistance?
- What governs the tradeoff between mutagenic and anti-mutagenic roles of MMR in TNR diseases?
- What occurs during collisions between DNA repair mechanisms with each other or other processes on DNA?
- What is the nature and origin of catastrophic mutational events?
- These questions are each complex in their own right and have remained difficult to answer using traditional techniques, but the SPORE assay provides a direct way to address each of them. The likely outcomes of my laboratory’s approach during the R35 award will be numerous breakthroughs in our understanding of genomic stability and how it can be manipulated in living cells; with a long-term impact being a sea-change in the ability to probe and exploit DNA damage repair mechanisms to treat disease.
Research that is reported in this post is supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R35GM133483. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
