Establishment of a Doxycycline-Inducible HERC1- and HERC2-Deficient Cell Line

Abstract

HERC proteins comprise RCC-like domain that presumably possesses Ran-GEF activity and a HECT domain that contains ubiquitin E3 ligase activity. HERC1 and HERC2 are giant proteins comprising more than 4800 amino acids, and are structurally similar to multiple RCC-like domains and a C-terminal HECT domain. We previously showed that HERC2 is an E3 that targets the familial breast and ovarian cancer susceptibility gene product BRCA1 for degradation, controls G2/M checkpoint of the cell cycle, and regulates DNA replication through interaction with Claspin. However, many functions of HERC2 remain to be clarified. To further investigate the HERC2 functions, we generated a specific antibody to HERC2, and established cell lines in which both HERC1 and HERC2 can be inhibited simultaneously, eliminating a possible compensation of HERC2 function by HERC1. We created a recombinant polypeptide encoding C-terminus of HERC2 containing the HECT domain and generated a rabbit polyclonal antibody to the polypeptide. We then established Doxycyclin (Dox)-inducible HERC2-defective cell lines from HeLa, HCT116 or U2OS cells by infecting lentivirus that is comprised of the CS-RfA-ETBsd-shHERC2 vector expressing blasticidin resistant gene, Tet repressor, and HERC2-specific shRNA under H1tetO promoter/operator regulation, after selection with blasticidin. Dox-inducible HERC1/HERC2 double-defective cell lines were then established by infecting a lentivirus with HERC1-specific shRNA and puromycin resistance to the HERC2-defective cell lines after double selection with blasticidin and puromycin. The successful simultaneous inhibition of HERC1 and HERC2 expression after Dox-treatment was verified by immunoblotting. The dual inhibition of HERC1 and HERC2 severely compromised the retention of Replication protein A (RPA) at ionizing irradiation-induced DNA damage loci whereas single inhibition of HERC1 or HERC2 demonstrated minimal effects. Hence, cell lines with Dox-inducible dual inhibition of HERC1 and HERC2 were established. The cells would contribute to further analyses of the roles of HERC1 and HERC2 in cell-cycles and DNA damage response.