RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.
Bottom Line: We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage.These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells.Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.
Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.Show MeSH
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Mentions: Next, we studied the impact of RAD51 overexpression and RAD54L+RAD54B depletion on cellular growth. RAD51 overexpression in S33 cells has previously been shown to decrease plating efficiency (6). To extend this observation, we monitored the ability of cells to perform DNA synthesis (i.e. to incorporate EdU) following changes in RAD51 and/or translocase levels. Cells pulsed with EdU for 45 min were subsequently quantified for EdU staining (Figure 5a). Both RAD51 overexpression and translocase depletion decreased the fraction of EdU-positive S33 cells about 1.5-fold (30 ± 4% and 28 ± 4%, respectively) relative to control cells (44 ± 4%). Furthermore, the combination of the two treatments resulted in a synergistic block to proliferation; the EdU-positive fraction was reduced 4.5-fold (10 ± 2%; P < 0.0005) compared to the control. In MCF7 cells, translocase depletion similarly resulted in a significant reduction in the EdU positivity (Figure 5a), with a striking 4.7-fold reduction at 72 h (10 ± 7% of translocase-depleted cells versus 47 ± 9% of control cells; P < 0.05). These results suggest that the accumulation of RAD51 complexes on undamaged chromosomes blocks growth.
Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.