Cell culture, reagents, and antibodies
HEK 293T and HCT116 cells were cultured in DME and McCoy's media supplemented with 10% fetal bovine serum, respectively. Commercially available anti-PCNA (PC10), anti-ubiquitin (P4D1), anti-polyubiquitin (FK2), anti-myc (9E10), anti-HA (12CA5), anti-FLAG (M2), anti-V5 antibodies, and anti-RAD18 (K-15) were used. Polyclonal anti-SHPRH antibody was previously described (Sood et al., 2003
). MMS, MMC, and mimosine were purchased from Sigma-Aldrich.
Construction of various expression plasmids
Full-length cDNA of human SHPRH was obtained by PCR with IMAGE clones (available from GenBank/EMBL/DDBJ under accession nos. AI888407 and AA255592) and RACE-PCR products as templates. cDNA encoding human RAD18, PCNA, MMS2 (UEV2), and RAD6 (HHR6B) were obtained from Mammalian Genome Collection (RAD18, MHS1010-52750; PCNA, MHS1011-58526; MMS2, MHS1011-62471; HHR6B, MHS1011-62750). Expression plasmids were constructed by subcloning each cDNA into pcDNA3.1-myc-His, p3XFLAG-CMV, or pGEX-6P-1. UBC13(wild type or C87A)-HA–expressing plasmids were gifts from D. Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany) and J. Kehrl (National Institute of Allergy and Infectious Diseases, Bethesda, MD), respectively. Point mutations in the RING finger domain of SHPRH(C1432A) and in PCNA(K164R) were introduced by using an in vitro mutagenesis method (QuikChange; Stratagene).
Coimmunoprecipitation and GST pull-down assays
For coimmunoprecipitation assay, HEK 293T cells were transfected with various combinations of expression plasmids using FuGENE 6 transfection reagent (Roche) and lysed in the TNE buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40, 1 mM EDTA, 8% glycerol, 0.5 mM DTT, 50 mM NaF, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). For the GST pull-down assay, various GST fusion proteins were expressed in Escherichia coli strain BL21 (DE3)-RIL (Stratagene) and purified using glutathione–Sepharose beads (GE Healthcare). 20 μg of GST fusion proteins were used for pulling down SHPRH-FLAG or RAD18-FLAG expressed in HEK 293T cells.
Generation of lentivirus-mediated stable knockdown cells
SHPRH-silencing vectors were constructed by cloning the target sequences of 5′-TTCAATGCCCTCCTACAC-3′ (construct B) and 5′-AGTGTCCATCCTTTCCAT-3′ (construct C) into the lentivirus-based expression vector pLL3.7 (a gift from V. Parijs, Massachusetts Institute of Technology, Cambridge, MA). RAD18-silencing lentivirus vector was purchased from Open Biosystems. Lentivirus packaging plasmids were gifts from F. Candotti (National Human Genome Research Institute, Bethesda, MD). Lentivirus-infected cells were selected by the expression of GFP (SHPRH) or by puromycin (RAD18). The expression level of SHPRH was examined by Western blot or by RT-PCR with primers 5′-GAGCAACTCTGATCATCTCTCCAAG-3′ and 5′-GATAGAGAAGTCGAACCCACCAGTG-3′. Primers used for amplifying ACTIN as a control were 5′-GCTCGTCGTCGACAACGGCTC-3′ and 5′-CAAACATGATCTGGGTCATCTTCTC-3′. SHPRH-silenced clones B2, B11 (construct B), and C4 (construct C) were used in this study.
Detection of MMS-induced PCNA polyubiquitination, sensitivity assay, and chromosome breakage analysis
HCT116 cells were treated with MMS for 2 h, washed with PBS, and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Triton X-100, 0.5 mM DTT, 50 mM NaF, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). In some experiments, cells were treated with 0.5 mM mimosine for 20 h. For measuring MMS sensitivity, SHPRH-knockdown cells were treated with increasing concentrations of MMS for 1 h, washed with PBS three times, replated onto 6-well plates at defined cell densities, and cultured for 10 d. Colonies grown from the surviving cells were counted and expressed as a survival fraction (%) compared with untreated cells as 100%. For analyzing MMS-induced chromosomal breaks, SHPRH-knockdown cells were treated with 0.01% MMS for 1 h, washed with PBS three times, and cultured for 24 h. At least 100 metaphase spreads from each clone were analyzed.
Online supplemental material
Fig. S1 shows moderate homology between the SWI2/SNF2 domains in SHPRH and yeast Rad5. Fig. S2 shows that SHPRH
expression in the yeast rad5
strain could not complement UV sensitivity. Fig. S3 shows that RAD18 and RAD6B induced exclusive monoubiquitination of PCNA, the reduced activity of SHPRH(ΔRING) for PCNA polyubiquitination, and that SHPRH did not promote polyubiquitination of c-JUN. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200606145/DC1