(Georgia Institute of Technology, 2017-05)
Gordon, Katherine
Double-strand breaks (DSBs) in DNA are detrimental, as they can cause mutations and genomic rearrangements, which in turn leads to cancer and other diseases. Recent research reveals that DSBs can be repaired by RNA-templated homologous recombination. However, RNA-templated repair of DSBs is not well understood. In order to better understand the mechanism of RNA-templated repair of DSBs, the current research aims to identify the proteins that facilitate the repair. The research utilizes a system wherein RNA-templated repair of DSBs is known to occur. A yeast overexpression plasmid library was produced in order to test the ability of fragments of the yeast genome to facilitate RNA-templated repair of a DSB when these are highly expressed in the yeast cells. In order to test the ability of added gene fragments to facilitate RNA-templated repair of DSBs, the experimental candidates were exposed to galactose in order to induce a DSB, to activate the transcription of the overexpressed gene fragment, and to initiate the transcription of anti-sense RNA used to repair the break. The candidates were then moved to medium without histidine in order to assess the frequency of repair. Once a large number of colonies (~50,000) are screened, we expect to identify several proteins that facilitate RNA-templated repair of DSBs. Identifying the specific genes that facilitate this repair mechanism will assist in characterizing the functionality of the RNA-templated DNA repair mechanism. Identifying these genes will also allow for better predictions for how this same phenomenon may occur in human cells.
(Georgia Institute of Technology, 2013-12-13)
Shetty, Lahari
Ribonucleotides can be incorporated into the yeast genome through a variety of mechanisms, including through DNA polymerazation, DNA priming, and oxidative damage. Ribonucleotides contain a reactive 2’ hydroxyl group on the sugar, which can distort the DNA double helix and lead to defective replication and transcription and ultimately mutagenesis. Ribonucleotide excision repair (RER) has been found to remove ribonucleotides through the enzyme RNase H2, though the in vivo substrate specificity is not known. Nucleotide excision repair (NER) removes bulky lesions formed in DNA, however its role in the extraction of ribonucleotides has not yet been determined in eukaryotes. Previously developed oligonucleotide-driven gene correction assays in Saccharomyces cerevisiae, or baker’s yeast, have shown that paired and mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and can result in a genetic alteration. We implemented this assay to examine whether RNase H2 and NER can target specific rNMPs in DNA. Our results deliver new evidence that RNase H2 specifically recognizes isolated paired and mispaired rNMPs embedded in yeast genomic DNA and that the NER mechanism can recognize an isolated paired rNMP as damage during DNA double-strand break repair in yeast.
(Georgia Institute of Technology, 2013-04)
Gombolay, Alli
Increasing evidence suggests that ribonucleotides may represent one of the most common non-standard nucleotides found in genomic DNA. Therefore, it is important to understand the extent to which ribonucleotides alter genomic integrity and the cellular mechanisms that are responsible for removing them. We developed oligonucleotide-driven gene correction assays in the yeast Saccharomyces cerevisiae to show that, if not removed, mispaired and paired ribonucleotides embedded in genomic DNA serve as templates for DNA synthesis and could cause genetic change. We found that RNase H type 2 targets single paired and mispaired ribonucleotides, as well as a stretch of two or three ribonucleotides embedded in DNA, and the nucleotide excision repair system can target single paired ribonucleotides as damage.