Breakfast Club Seminar Series
"Mechanisms of RNA-driven DNA Modification and Repair"
Francesca Storici, PhD
School of Biology
As we have recently demonstrated in yeast Saccharomyces cerevisiae cells, short ribonucleoside monophosphate (rNMP) tracts carried within DNA oligonucleotides (oligos) can serve as templates for DNA synthesis at the chromosomal level and transfer a genetic change. The capacity of RNA to directly modify genomic DNA was also found in the bacterium Escherichia coli and in human embryonic kidney (HEK-293) cells, thus suggesting that the phenomenon is conserved. These findings stimulated two major avenues of research: i) to study how scattered ribonucleotides embedded in DNA can affect genome stability, and ii) how endogenous RNA transcripts can directly drive DNA modifications in cells.
With the goal of understanding to what extent rNMPs embedded in DNA may alter genome integrity and what factors affect their stability we developed oligo-driven gene correction assays in E. coli and S. cerevisiae. We showed that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and can produce a genetic change. We demonstrated that RNase H type 2 can recognize single mispaired rNMPs embedded in DNA at the chromosomal level in both E. coli and S. cerevisiae cells. We found that the mismatch repair (MMR) system can remove mispairs of scattered rNMPs embedded in DNA both in yeast and E. coli cells and that instead a mismatch within an RNA-DNA heteroduplex region required RNase H type 1 for removal. Our data revealed overlapping activity between the MMR system and RNase H type 2 in processing the RNA/DNA mispairs. In the absence of mismatch repair and RNases H, ribonucleotide-driven gene modification increased by a factor of ~50 in yeast and 77,000 in E. coli.
In order to determine the capacity of an RNA transcript to directly serve as a template for chromosomal DNA modifications, we developed two assays in yeast. An RNA transcript was tested to repair a chromosomal double-strand break (DSB) generated either in the DNA from which the transcript itself originated or in an ectopic DNA locus homologous to the transcript. Data will be shown revealing the capacity of an RNA transcript to efficiently repair a DSB in its own DNA.
- Workflow Status: Published
- Created By: Colly Mitchell
- Created: 07/16/2012
- Modified By: Fletcher Moore
- Modified: 10/07/2016