The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells
Figure 1
The proteasome inhibitor MG132 decreases HR in HeLa cells. (a) HR substrate in HeLaG1 cells. HeLaG1 cells contain an intrachromosomal recombination substrate consisting of two hygromycin resistance (hygR) alleles inactivated by the insertion of 10 bp and separated by the NeoR that confers resistance to G418. One hygR allele is inactivated at the PvuI site, whereas the other one is inactivated at the SacII site. An intrachromosomal recombination event leads to the reversion to hygromycin resistance phenotype (HYGR) by gene conversion or reciprocal exchange. (b) MG132 reduces HR in HeLa cells. Cells were seeded in a 6-well plate at the concentration of per well. After 24 h cells were incubated for 5 hours with 0.3% DMSO as control or with 3 μM, 10 μM, and 30 μM MG132. Next, cells were trypsinized, counted, and seeded as described in Material and Methods. After 24 hours, hygromycin (200 μg/mL) was added. After 10 days, when the clones were visible, plates were stained with crystal violet and hygromycin-resistant clones were counted. The frequency of HR was calculated by dividing the total number of HYGR colonies by the number of viable cells. Data are reported as mean of five independent experiments ± standard deviation. Results are statistically analyzed using Student’s -test. .
