DHX8對DNA雙股斷裂點招募RNA聚合酶II的影響

Abstract

當DNA結構受到外部或內在產生的有毒因素時（例如：紫外線作為外源毒性或R-loop的形成視為內源因素），會對genomic DNA中造成DNA損傷。如果未能即時修復這些DNA損傷，最終可能會導致細胞的衰亡，也因此細胞發展了各種機制來維護DNA結構的完整性。R-loop是由單股DNA和DNA-RNA hybrid所組成，而R-loop的生成會使DNA結構的不穩定，也因此R-loop的解開是被需要的。 DHX8 是RNA helicase，本身作為splicing factor可以從spliceosome上釋出mRNA而聞名。先前研究中已經證實DHX8在體外可以解開DNA-RNA hybrid，且我們發現DHX8 會影響R-loop的累積。此外，我們先前的研究亦有發現DHX8參與了ATR-Chk2 repair pathway，並且在缺乏DHX8的細胞中，造成RPA無法累積在 DNA 斷裂處。 然而，我們的最近結果顯示出：當加入DNA 損傷藥劑CPT 時，DHX8對於消除double-strand break（DSB）上的DNA-RNA hybrid是沒有效用的。此外，我們意外的發現：加入DNA 損傷藥劑CPT，在缺乏DHX8的情況下，會無法累積在DNA斷裂處上的DNA-RNA hybrid。 因前人的研究指出RNA polymerase（RNAPII）可以轉錄DNA-induced long non-coding RNA（dilncRNA），且該RNA可以參與DNA損傷（DDR）的啟動。基於上述的研究，我們推測DHX8與召集RNAPII到DSB上具有潛在作用，我們的結果顯示出：在缺乏DHX8的細胞中，RNAPII被召集至DSB的數量明顯減少，這說明DHX8在促進RNAPII被召集到DSB中發揮了關鍵作用。

Once the DNA structure is challenged by the toxic external or internal factors, such as ultraviolet light as exogenous toxicity or the formation of R-loops as an endogenous factor, it can result in the generation of DNA lesions within the genomic DNA. Failure to repair these DNA lesions can ultimately lead to cell death. Therefore, cells have developed various mechanisms to preserve genomic integrity. R-loop is a structure composed of single-strand DNA and DNA-RNA hybrid. The formation of R-loops destabilizes the DNA structure, and therefore is required to be unwound. DHX8, an RNA helicase known for mRNA releasing from spliceosome, has been demonstrated to unwind DNA-RNA hybrid in vitro. Our previous study showed that DHX8 depletion leads to accumulation of genomic R-loop, and inhibits the activation of the ATR-Chk1 repair pathway. Depletion of DHX8 displays a defect of replication protein A (RPA) recruitment to DNA damage sites, thereby impeding the further steps of the DNA repair pathway. Surprisingly, our results demonstrate that DHX8 depletion fails to accumulate DNA-RNA hybrids at double-strand break (DSB) sites upon camptothecin (CPT) treatment. We show that DHX8 is associated with RNA polymerase II (RNAPII), R-loops and DNA breaks induced by CPT. Recent studies indicate that RNAPII can transcribe DNA damage-induced long non-coding RNAs (dilncRNAs), which are involved in initiating DNA damage response (DDR). To test the potential involvement of DHX8 in the recruitment of RNAPII to double-strand breaks (DSBs), proximity ligation assay (PLA) for RNAPII and 𝛾H2AX was performed to detect RNAPII at DSBs. The result shows that depletion of DHX8 leads to a decrease of RNAPII at DSB sites, suggesting that DHX8 plays a critical role in regulating the recruitment of RNAPII to DSBs, and this recruitment is important for the initiation of DNA repair.