Aquarius is required for proper CtIP expression and homologous recombination repair

Ryo Sakasai,Mitsuo Wakasugi,Mayu Isono,Tsukasa Matsunaga,Mitsumasa Hashimoto,Yumi Sunatani,Kuniyoshi Iwabuchi,Atsushi Shibata,Tadashi Matsui

doi:10.1038/s41598-017-13695-4

Ryo Sakasai, Mitsuo Wakasugi + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-13695-4

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Abstract

Accumulating evidence indicates that transcription is closely related to DNA damage formation and that the loss of RNA biogenesis factors causes genome instability. However, whether such factors are involved in DNA damage responses remains unclear. We focus here on the RNA helicase Aquarius (AQR), a known R-loop processing factor, and show that its depletion in human cells results in the accumulation of DNA damage during S phase, mediated by R-loop formation. We investigated the involvement of Aquarius in DNA damage responses and found that AQR knockdown decreased DNA damage-induced foci formation of Rad51 and replication protein A, suggesting that Aquarius contributes to homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Interestingly, the protein level of CtIP, a DSB processing factor, was decreased in AQR-knockdown cells. Exogenous expression of Aquarius partially restored CtIP protein level; however, CtIP overproduction did not rescue defective HR in AQR-knockdown cells. In accordance with these data, Aquarius depletion sensitized cells to genotoxic agents. We propose that Aquarius contributes to the maintenance of genomic stability via regulation of HR by CtIP-dependent and -independent pathways.

Highlights

Cells harbour several systems to counter various types of DNA damage, including DNA double-strand breaks (DSBs) and DNA replication stress, which trigger DNA damage responses (DDRs) such as DNA repair, cell cycle arrest, and apoptosis
These 53BP1 foci were mainly observed in cyclin A-positive cells, suggesting that the underlying damage arises through DNA replication (Fig. 1b). This 53BP1 foci formation was suppressed by expression of siRNA-resistant GFP-tagged wild-type (WT) Aquarius and a helicase-dead (HD) mutant (Y1196A)[22] (Fig. 1c,d), as well as by GFP-RNase H1 overproduction (Fig. 1e). These results suggest that spontaneous DNA damage accumulates through collisions with DNA replication forks and R-loops in Aquarius-depleted cells
This R-loop formation and subsequent DNA damage were considered to be derived from the failure of splicing because Aquarius is a pre-mRNA splicing factor, but, interestingly, Aquarius helicase activity was not necessary to prevent the accumulation of spontaneous DNA damage

Summary

Introduction

Cells harbour several systems to counter various types of DNA damage, including DNA double-strand breaks (DSBs) and DNA replication stress, which trigger DNA damage responses (DDRs) such as DNA repair, cell cycle arrest, and apoptosis. Dysfunction of RNA export factors or pre-mRNA splicing factors causes transcription-associated DNA damage, leading to hyper-recombination in yeast and human cells[11,12,13,14,15]. In the absence of Aquarius, accumulated R-loops are resolved in pathways that are dependent on the transcription-coupled nucleotide excision repair (TC-NER) factor CSB (Cockayne syndrome group B) and the NER endonuclease XPF (xeroderma pigmentosum group F)[21]. Aquarius depletion causes CtIP downregulation and sensitizes cells to DNA-damaging agents. These results suggest that Aquarius is required for the maintenance of genome integrity via CtIP-dependent and -independent pathways

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Conclusion