RNA-mediated double-stranded break repair in mammalian cells
This study seeks to examine the capability of RNA to introduce mutations into genomic DNA, and to provide insight into the enzyme(s) responsible for this phenomenon. To do this, a double-stranded break is induced in HEK 658D cells, and oligonucleotides containing RNA tracts with homology to the area around the break are introduced to examine if they can mediate repair of this area. In the first part of the study, oligonucleotides are introduced that contain a single-nucleotide substitution at the site of the break, and if the RNA is used as a template for repair, then this nucleotide should be incorporated into the genome of the cell. Oligonucleotides are introduced that contain either all-DNA, or DNA with a 6-nucleotide RNA tract at the break. These two classes of oligonucleotides are homologous to either the forward or reverse strands of the area around the break. This allows us to examine if there is a strand bias for repair, and if that bias is the same for DNA- and RNA-mediated repair, which will provide clues into the enzyme involved in repair.