Ribonucleotides, also known as ribonucleoside monophosphates (rNMPs), are the most abundant non-canonical nucleotides incorporated into genomic DNA. Despite the relevance, information about their repair pathways, consequences, and profiles is still lacking. Exploiting the use of oligonucleotides containing rNMPs in a molecular approach to generate various RNA/DNA hybrids of chosen sequence and structure at the chromosomal level in cells, we show that mispaired rNMPs embedded into genomic DNA are not only targeted by ribonucleases H (RNases H) but also by the mismatch repair (MMR) system both in E. coli and S. cerevisiae cells. In addition, we discovered that paired rNMPs in DNA are targets of both RNase H type 2 and nucleotide excision repair (NER) in yeast. Also, we report atomic force microscopy (AFM)-based single molecule elasticity measurement, molecular dynamics simulation, and nuclear magnetic resonance spectroscopy results, showing that rNMPs in short DNA duplexes can change the elastic and structural properties of DNA. Lastly, we developed ribose-seq, a method for capturing rNMPs embedded in DNA. High-throughput sequencing of rNMP-captured molecules from the yeast S. cerevisiae revealed widespread but non-random rNMP distribution with preferences in base composition of rNMPs and neighboring DNA sequence context in both nuclear and mitochondrial DNA. With ribose-seq, systematic profiling of rNMP incorporation into genomic DNA is achieved, potentially allowing determination of specific signatures of rNMPs in DNA which could help to better understand the nature of rNMP repair mechanisms, effect of rNMPs on DNA mechanical properties and structure, and eventually rNMP impact on genome integrity.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/53534 |
| Date | 08 June 2015 |
| Creators | Koh, Kyung Duk |
| Contributors | Storici, Francesca |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
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