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School of Biological Sciences

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search.filters.applied.f.advisor: Storici, Francesca × End date: 2013 × Start date: 2010 ×

Publication Search Results

Now showing 1 - 5 of 5
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Ribonucleotides in yeast genomic DNA are targets of RNase H2 and nucleotide excision repair

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.

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Ribonucleotides embedded in yeast genomic DNA are targets of RNase H2 and the nucleotide excision repair system

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.

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Protein-assisted targeting of genes in yeast and human cells

2013-06-28 , Ruff, Patrick

This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for gene targeting to have that double-stranded DNA on the 5’ side of our targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering targeting DNA within close proximity to the site of modification, gene targeting frequencies can be increased.

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Studies on the mechanisms of RNA-driven DNA repair and modification

2011-11-14 , Shen, Ying

Our previous studies have demonstrated that RNA can serve as a template for double-strand break (DSB) repair in the yeast Saccharomyces cerevisiae using synthetic RNA-containing oligonucleotides (oligos). Following this initial work, we show that the RNA tract of RNA-containing oligos can be copied into DNA to transfer a genetic change at the chromosomal level also in the bacterium Escherichia coli and in human cells. Exploiting the use of oligos containing ribonucleoside monophosphates (rNMPs), we developed a molecular approach to generate RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in both yeast and E. coli cells. Such technique allows us to study how rNMPs present in the DNA genome of cells are tolerated by cells, what factors recognize and target rNMPs in DNA and to what extent the embedded rNMPs may alter genome integrity. Here we proved that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis during chromosomal replication and produce a genetic change. We discovered that mispaired rNMPs that are embedded in genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in yeast and in E. coli. Our data reveal novel substrates for the MMR system, and also uncover an unpredicted competition between RNase H and MMR for the RNA/DNA mispairs.

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Gene targeting at and distant from DNA breaks in yeast and human cells

2013-04-02 , Stuckey, Samantha Anne

Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.

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