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.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/45969 |
| Date | 14 November 2011 |
| Creators | Shen, Ying |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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