Dr. David S. Pederson
Nucleosome Dynamics & Repair of DNA Damage in Chromatin
The Pederson lab studies the repair of oxidatively damaged DNA in chromatin. Both normal oxidative metabolism and ionizing radiation create reactive oxygen species that can damage DNA. Some of the resulting DNA lesions are cytotoxic, while others are mutagenic, and may thereby increase the risk of cancer. Most of the ~10 trillion oxidative lesions that form every second (!!) in the average human are repaired by Base Excision Repair (BER) enzymes. We know a great deal about how these enzymes repair lesions in naked DNA, but much less about how they function in cells, where access to DNA is restricted by its packaging in chromatin. Our research goals are to  discover and elucidate mechanisms that facilitate BER in chromatin, and  to identify BER enzyme variants that increase cancer risk, due to their failure to function normally in chromatin.
Mechanisms that facilitate BER in chromatin. Most of the DNA in chromatin is packaged in nucleosomes. DNA in nucleosomes is wrapped around a histone protein core, which often restricts the binding of transcription and DNA replication and repair factors. Cells possess histone chaperones and chromatin-remodeling agents that help some of these factors bind to their DNA targets. However, there is as yet no direct evidence that these remodeling agents act at sites of BER, and our in vitro studies indicate that a significant fraction of the oxidative lesions that form in nucleosomes can be repaired in the absence of chromatin-remodeling agents. We determined as well that this repair occurs without irreversibly disrupting the host nucleosome, and within a succession of ternary complexes that assemble with lesion-containing nucleosomes (Prasad et al., 2007; Odell et al., 2011; Cannan et al., in revision). Periodic partial unwrapping of nucleosomal DNA enables BER enzymes to bind lesions that are occluded in intact nucleosomes. To determine if spontaneous unwrapping could account for the relatively fast repair of oxidized bases in vivo, we have conducted pre-steady state kinetic analyses, to measure the frequency with which oxidized bases are exposed by unwrapping (Maher et al., 2013). These studies suggested that there are additional agents or mechanisms that promote BER in cells. To determine how DNA glycosylases discover oxidative lesions in vivo, in a sea of undamaged DNA, we measured the cellular concentration of two DNA glycosylases, along with their specific and non-specific DNA binding constants (Odell et al., 2010). For one of the two glycosylases, these parameters were consistent with a mechanism in which the enzyme binds DNA non-specifically, and then scans along DNA until it encounters an oxidative lesion. To further test this hypothesis, we are collaborating with our UVM colleagues, Susan Wallace and David Warshaw, to visualize interactions between BER factors and chromatin substrates, using single molecule technologies.
BER pathologies and genome instability. Exposure to ionizing radiation often leads to multiple, tightly clustered oxidative lesions in DNA. In vitro studies indicated that attempted BER of closely opposed oxidative lesions can produce double strand breaks in DNA, which may be cytotoxic or subject to error-prone repair that increases the risk of mutation. We demonstrated that nucleosomes help protect DNA from double strand DNA breaks during BER of clustered lesions (Cannan et al., 2014; Cannan et al., 2016). Certain mutations in BER enzymes can produce repair defects that increase the risk of cancer. In collaborative studies with Joann Sweasy, we recently identified a rare germline variant of a BER enzyme, which functions normally in biochemical assays but promotes genome instability when expressed in human cells in culture. We currently are investigating this and selected other enzyme variants, as we expect them to reveal new information about the in vivo function and regulation of BER enzymes.
Structural transitions among nucleosomes that package DNA in eukaryotes help regulate transcription, DNA replication and repair.
BER enzymes can directly bind lesions in nucleosomes if they are optimally oriented relative to the underlying histone octamer.
Cannan WJ, Pederson DS. Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin J Cell Physiol. 2016 Jan;231(1):3-14
Cannan WJ, Tsang BP, Wallace SS, Pederson DS. Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages J Biol Chem. 2014 Jul 18;289(29):19881-93
Odell ID, Wallace SS, Pederson DS. Rules of engagement for base excision repair in chromatin J Cell Physiol. 2013 Feb;228(2):258-66
Maher RL, Prasad A, Rizvanova O, Wallace SS, Pederson DS. Contribution of DNA unwrapping from histone octamers to the repair of oxidatively damaged DNA in nucleosomes DNA Repair (Amst). 2013 Nov;12(11):964-71
Odell ID, Barbour JE, Murphy DL, Della-Maria JA, Sweasy JB, Tomkinson AE, Wallace SS, Pederson DS. Nucleosome disruption by DNA ligase III-XRCC1 promotes efficient base excision repair Mol Cell Biol. 2011 Nov;31(22):4623-32