Role of DVC1 in repair of nitrogen mustard-induced DNA protein crosslink in human bronchial epithelial cells

Abstract

Objective To investigate the effect of DNA damage protein targeting VCP (DVC1) in DNA protein crosslink repair induced by nitrogen mustard in human bronchial epithelial cells (16HBE). Methods DVC1 siRNA was used to interfere its expression in 16HBE. Then the cells were exposed to 50 μmol/L nitrogen mustard, and the expression levels of DNA damage repair proteins were detected by Western blotting. The total content of DNA-protein crosslink was determined by fluorescence microplate analyzer from 1 to 24 h. The crosslink content of DNA with O6-methylguanine-DNA methyltransferase (MGMT), a methyltransferase, was detected by Slot blotting. The level of ubiquitinated MGMT was detected by immunoprecipitation. After GST-DVC1 fusion protein was constructed, GST pull-down was used to detect the binding of DVC1 with MGMT and ubquitin. Results After si-DVC1 intervention, the total content of DNA-protein crosslink was increased, and exposure to nitrogen mustard decreased the level of prototype of MGMT, but increased the level of ubiquitinated MGMT. DVC1 could bind to ubquitin, and si-DVC1 intervention further enhanced the ubiquitination of MGMT, as well as the crosslink between MGMT and DNA, which was similar with NSC697923 (an E2 ubiquitin-conjugating inhibitor) and MG132 (a proteasome inhibitor) treatment. The expression of Rad51 and Ku70, 2 DNA repair proteins, were increased and decreased respectively by si-DVC1 interference. Conclusion Clearance of DNA-protein crossslink is depend on ubiquitination of MGMT. DVC1 promotes the clearance of nitrogen mustard induced DNA-protein crossslink by interacting with ubiquitin, and maintain the ability of homologous recombination repair.

Keywords

• nitrogen mustard
• dvc1
• dna-protein crosslink o6-methylguanine-dna methyltransferase
• human bronchial epithelial cells