Abstract
Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. We recently reported a signaling pathway induced upon alkylation damage to recruit the AlkB homolog 3, α-ketoglutarate-dependent dioxygenase (ALKBH3)-activating signal cointegrator 1 complex subunit 3 (ASCC3) dealkylase-helicase repair complex. As in other DDR pathways, the recruitment of these repair factors is mediated through a ubiquitin-dependent mechanism. However, the machinery that coordinates the proper assembly of this repair complex and controls its recruitment is still poorly defined. Here, we demonstrate that the ASCC1 accessory subunit is important for the regulation of ASCC complex function. ASCC1 interacts with the ASCC complex through the ASCC3 helicase subunit. We find that ASCC1 is present at nuclear speckle foci prior to damage, but leaves the foci in response to alkylation. Strikingly, ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2. These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Consistent with its role in alkylation damage signaling and repair, ASCC1 knockout through a CRISPR/Cas9 approach results in alkylation damage sensitivity in a manner epistatic with ASCC3. Together, our results identify a critical regulator of the ALKBH3-ASCC alkylation damage signaling pathway and suggest a potential role for RNA-interacting domains in the alkylation damage response.
Highlights
Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors
We find that ASCC1, unlike ASCC2 or activating signal cointegrator complex subunit 3 (ASCC3), is constitutively present at nuclear speckle foci, yet it is removed from these nuclear regions upon alkylation damage
We focused on ASCC1, a protein previously shown to copurify with ASCC2 and ASCC3 [17, 18]
Summary
Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2 These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Because of the diverse chemical nature of alkylation damage, multiple pathways have evolved to protect the genome from alkylation damage These include base-excision repair (BER), direct reversal by O6-methylguanine methyltransferase (MGMT), and the AlkB family of demethylases/dealkylases [1, 2, 4]. Our data suggest a novel regulatory mechanism for the ALKBH3–ASCC repair pathway wherein ASCC1 modulates the localization and function of the complex components
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