-
[show abstract]
[hide abstract]
ABSTRACT: Numerous studies have shown that ribonucleoside monophosphates (rNMPs) are probably abundant among all nonstandard nucleotides occurring in genomic DNA. Therefore, it is important to understand to what extent rNMPs may alter genome integrity and what factors affect their stability. We developed oligonucleotide-driven gene correction assays in Escherichia coli and Saccharomyces cerevisiae to show that mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and produce a genetic change. We discovered that isolated mispaired rNMPs in chromosomal DNA are removed by the mismatch repair system in competition with RNase H type 2. However, a mismatch within an RNA-DNA heteroduplex region requires RNase H type 1 for removal. In the absence of mismatch repair and RNases H, ribonucleotide-driven gene modification increased by a factor of 47 in yeast and 77,000 in E. coli.
Nature Structural & Molecular Biology 12/2011; 19(1):98-104. · 12.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNA-containing oligonucleotides (oligos), RNA can serve as a template for DNA synthesis at the chromosomal level during the process of double-strand break (DSB) repair. Herein we show that the phenomenon of RNA-mediated DNA modification and repair is not limited to yeast cells. A tract of six ribonucleotides embedded in single-strand DNA oligos corresponding to either lagging or leading strand sequences could serve as a template to correct a defective lacZ marker gene in the chromosome of the bacterium Escherichia coli. In order to test the capacity of RNA to modify DNA in mammalian cells, we utilized DNA oligos containing an embedded tract of six ribonucleotides, as well as oligos mostly made of RNA. These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells. We show that these RNA-containing oligos can serve as templates to repair a DSB in human cells and can introduce base changes into genomic or plasmid DNA. In both E. coli and human cells, the strand bias of chromosomal gene correction by the single-strand RNA-containing oligos was the same as that obtained for the corresponding DNA molecules. Therefore, the RNA-containing oligos are not converted into a cDNA before annealing with complementary DNA. Overall, we demonstrate that in both bacterial and human cells, as in yeast, RNA sequences can have a direct role in DNA genetic modification and remodeling.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2011; 717(1-2):91-8. · 2.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The discovery of RNA-templated DNA repair has revealed a novel case where genetic information can flow directly from RNA to genomic DNA without passing through a reverse transcript intermediate. As initially demonstrated in the yeast Saccharomyces cerevisiae via transformation by RNA-containing oligonucleotides (oligos), RNA sequences can serve as templates for chromosomal double-strand break (DSB) repair. Synthetic oligos containing embedded RNA tracts of various sizes, or even RNA-only molecules, although with lower efficiency, can guide DNA repair synthesis at sites of broken DNA. Mechanisms and circumstances in which cells can use RNA to repair DNA damage such as a DSB are yet to be identified. Here we show the approach we utilize to detect repair of a chromosomal DSB by RNA-containing oligos in yeast cells.
Methods in molecular biology (Clifton, N.J.) 01/2011; 745:193-204.
-
[show abstract]
[hide abstract]
ABSTRACT: Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.
Journal of Visualized Experiments 01/2010;
