Transcript-RNA-templated DNA recombination and repair

Abstract

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.

DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

“…However, recent evidence suggests that novel DSB repair pathways involving RNA templates may be functional throughout the cell cycle, including G 1 . RNA template-guided DSB repair has been reported in both mammalian cells and bacteria and involves RNA molecules that serve as templates to guide DNA synthesis during DSB repair (72)(73)(74)(75)(76). Regulation of RNA-DNA duplexes, including both their formation and resolution, may be critical to this repair process.…”

Mechanism of siRNA production from repetitive DNA

“…Thus, siRNA may target the HR machinery to the DSB to promote HR. On the other hand, it was recently shown in yeast that RNA transcripts can mediate HR of chromatin (Keskin et al 2014). Thus, QDE-2 may also target RNA transcripts made from the repetitive region to regulate their role in HR.…”

Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

“…For example, R-loops have long been associated with replicative stress and genome instability, whereas more recent work indicates that RNA-DNA hybrids can also promote DNA repair by mechanisms that remain to be elucidated (23)(24)(25)(26)(27). Considering the importance of RNA-DNA structures in DNA repair and genome instability, we proceeded to examine whether Polθ-helicase unwinds RNA-DNA duplexes with similar efficiency.…”