Lysine 40 acetylation of α tubulin (Ac-α tubulin) catalyzed by acetyltransferase αTAT1, marks stabilized microtubules. Recently, there is growing evidence to suggest the crosstalk between DNA damage response (DDR) and microtubule organization, we therefore investigated whether αTAT1 is involved in DDR. Following treatment with DNA damaging agents, increased levels of Ac-α tubulin were detected. We also observed significant induction of Ac-α tubulin after depletion of DNA repair proteins, suggesting that αTAT1 is positively regulated in response to DNA damage. Intriguingly, αTAT1 depletion decreased DNA damage-induced RPA phosphorylation and foci formation. Moreover, DNA damage -induced cell cycle arrest was significantly delayed in αTAT1-depleted cells, indicating defective checkpoint activation. The checkpoint defects by αTAT1 deficiency were restored by expression of wild-type αTAT1, but not by D157N αTAT1 (catalytically inactive αTAT1), indicating that the role of αTAT1 in the DDR is dependent on enzymatic activity. Furthermore, αTAT1-depleted DR-GFP U2OS cells resulted in a significant decrease in the frequency of homologous recombination repair. Collectively, our results suggest that αTAT1 may play an essential role in DNA damage checkpoints and DNA repair through its acetyltransferase activity.

The deubiquitinating enzyme USP1 contains highly conserved motifs forming its catalytic center. Recently, the COSMIC mutation database identified a mutation in USP1 at Asp‐199 in endometrial cancer. Here, we investigated the role of Asp‐199 for USP1 function. The mutation of aspartic acid to alanine (D199A) resulted in failure of USP1 to undergo autocleavage and form a complex with ubiquitin, indicating D199A Usp1 is catalytically inactive. The D199A mutation did not affect the interaction with Uaf1. Moreover, D199A Usp1 had defects in deubiquitination of FANCD2 and PCNA and displayed reduced FANCD2 foci formation and DNA repair efficiency. Furthermore, mutation of Asp‐199 to glutamic acid resulted in phenotypes similar to the D199A mutation. Collectively, our findings demonstrate the importance of Asp‐199 for USP1 activity and suggest the implications of USP1 downregulation in cancer.