Homologous recombination (HR) is a molecular process that plays multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life1. Generally, HR involves exchange of genetic information between two identical or nearly identical DNA molecules1; however, HR can also occur between RNA molecules, as shown for RNA viruses2. Previous research showed that synthetic RNA oligonucleotides (oligos) can template DNA double-strand break (DSB) repair in yeast and human cells3,4, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements5. Here we report that endogenous transcript RNA mediates HR with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect events of HR initiated by transcript RNA following repair of a chromosomal DSB occurring either in a homologous but remote locus (in trans), or in the same transcript-generating locus (in cis) in reverse transcription defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases (RNases) H1 and H2. In the presence of RNases H, DSB repair proceeds through a cDNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in cis facilitates Rad52-driven HR during DSB repair. In accord, we demonstrate that yeast and human Rad52 proteins efficiently catalyze annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of HR and DNA repair templated by transcript RNA. Thus, considering the abundance of RNA transcripts in cells, the impact of RNA on genomic stability and plasticity could be vast.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52’s biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.