Abstract
It was recently shown that the 5’ to 3’ exoribonuclease XRN2 is involved in the DNA damage response. Importantly, loss of XRN2 abrogates DNA double stranded break repair via the non-homologous end-joining pathway. However, the mechanistic details of how XRN2 functions in the non-homologous end-joining repair process are unknown. In this study, we elucidated that XRN2-mediated RNA:DNA hybrid resolution is required to allow Ku70 binding to DNA ends. These data suggest that XRN2 is required for the initiation of non-homologous end-joining repair. Interestingly, we uncovered a role for XRN2 in the homologous recombination repair pathway. Loss of XRN2 lead to a decrease in the repair of double strand breaks by homologous recombination. Strikingly, when we removed RNA:DNA hybrids by RNaseH1 over-expression, homologous recombination was not restored. We found RNA:DNA hybrid formation at and downstream of the DSB site, suggesting that unregulated transcription inhibits homologous recombination repair. In summary, our results indicate a relation between RNA:DNA hybrid resolution and double strand break repair pathway choice.
Highlights
IntroductionThe cell encounters a variety of different types of genomic lesions on a daily basis, and the most dangerous lesion is a DNA double stranded break (DSB), as one unrepaired DSB can be detrimental to cellular survival [1]
Maintaining genomic integrity over multiple rounds of replications is essential for cellular survival.The cell encounters a variety of different types of genomic lesions on a daily basis, and the most dangerous lesion is a DNA double stranded break (DSB), as one unrepaired DSB can be detrimental to cellular survival [1]
The EJ5 reporter cells measure total end-joining events; classical NHEJ (cNHEJ) and alternative NHEJ (aNHEJ) events and contain an expression cassette for green fluorescent protein (GFP) that is interrupted by an I-SceI endonuclease restriction site
Summary
The cell encounters a variety of different types of genomic lesions on a daily basis, and the most dangerous lesion is a DNA double stranded break (DSB), as one unrepaired DSB can be detrimental to cellular survival [1]. This DSB will initiate a signaling cascade known as the DNA damage response (DDR) [1] Unrepaired DSBs are dangerous due to the potential of the free DNA end to invade a neighboring DNA strand resulting in translocations which can contribute to disease states such as cancer [1]. It is commonly observed that highly transcribed areas of the genome preferentially employ the HR repair pathway [4], while transcriptionally silent areas of the genome tend to elicit the NHEJ repair pathway [5]
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