DNA Double-Strand Break in an Actively Transcribed Gene Is Accurately Repaired By Transcript-RNA in Cis Via a Homologous Recombination Mechanism

Cells are consistently bombarded from a variety of endogenous and exogenous DNA damaging sources, threatening genome stability. To maintain genome integrity, cells have devised pathways to respond to DNA damage. One of the most threatening forms of DNA damage is a double-strand break (DSB). Three major DSB repair mechanisms are known: non-homologous end joining (NHEJ), in which the broken ends of DNA are ligated together, often resulting in short deletions or insertions, microhomology-mediated end joining (MMEJ), which exploits microhomologous sequences to align the broken ends before joining, resulting in deletions flanking the DSB, and homologous recombination (HR), which has higher fidelity of repair requiring an intact donor DNA molecule with extensive homology to the broken DNA ends. With the advent of CRISPR RNA-guided DNA endonuclease enzymes, there is marked interest in understanding these pathways to facilitate accurate genome engineering events.

Recently, we showed that RNA can serve as a template for DSB repair indirectly, in the form of complementary DNA (cDNA), or directly, in the form of transcript-RNA in budding yeast (Keskin et al., Nature 2014; Meers et al., DNA Repair 2016). To better understand the mechanism of RNA-templated DSB repair, we constructed a yeast system in which an HO endonuclease generated DSB is induced in a constitutively transcribed marker DNA gene controlled by the strong promoter pTEF, similar to a break in an actively transcribed gene. In this constitutive system, direct repair by RNA is remarkably dominant compared to repair by cDNA. The frequency of RNA-templated DSB repair in this constitutive system is strongly reduced in the absence of the HR gene RAD52. Interestingly, deletion of genes associated with NHEJ (KU70, LIG4, or POL4) increases the frequency of DSB repair by transcript-RNA with 7-10% of cells surviving the DSB in a ku70 background being repaired by RNA. RNA-templated repair is stimulated by overexpression of reverse transcriptase. Furthermore, we find that functional mismatch repair (MMR) mechanism is required for efficient RNA-templated DSB repair, possibly because it prevents MMEJ. Overall, these results support an HR type of repair mechanism with donor transcript-RNA at a DSB site in constitutively expressed DNA.

We acknowledge support from the GAANN fellowship to C.M P200A150130-16, NIH grant GM115927, NSF grant MCB-1615335, and the HHMI Faculty Scholar grant #55108574 to F.S.