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: Studies (60,61) have established that yeast Rad54 has the fundamental biochemical activity of disassembling yeast Rad51-dsDNA filaments. However, evidence for the human RAD54 disassembly of RAD51 filaments in vitro remained to be demonstrated. To this end, we used the modified electrophoretic mobility shift assay previously employed with the yeast system (60,61). In this assay (Figure 4a), Rad51 filaments are formed on radiolabeled dsDNA and then excess unlabeled ‘scavenger’ dsDNA is added to bind any Rad51 that disassembles from the original nucleoprotein filaments and thus prevent their reassembly on the original dsDNA. As shown in Figure 4b, yeast Rad51-dsDNA filaments enter the gel as a discrete species and are disassembled by Rad54 in a time-dependent manner to produce freely migrating dsDNA. Under the same magnesium-ATP conditions, human RAD51–dsDNA complexes do not enter the gel and remain in the well as aggregated species. It is not clear if these represent individual RAD51 filaments, complex filament networks or nonspecific DNA co-aggregates. RAD51-ssDNA filaments formed under similar conditions are unstable and are not productive for DNA strand exchange, while the addition of calcium ions inhibits RAD51 ATP hydrolysis and allows formation of extended, stable filaments (62). When calcium is included in the reactions (Figure 4b, c), RAD51-dsDNA filaments now enter the gel as a discrete species, and these filaments show very little turnover as evident by a lack of increase in the level of free substrate with time. The addition of RAD54 causes the nucleoprotein complexes to supershift into the wells. While the disassembly of RAD51-dsDNA filaments is inefficient at higher calcium concentrations (2 mM), titrating down the calcium concentration allows a progressive increase in the amount of free dsDNA that is liberated by RAD54 in a time-dependent manner (Figure 4c). This behavior is analogous to experiments with yeast proteins, where both the Rad54 and Rad51 ATPase activities are required for optimal disassembly of Rad51-dsDNA filaments (61). To test the possibility that RAD54 might also cause filament disassembly on ssDNA, we performed the assay under the low calcium (0.5 mM) condition and formed RAD51 filaments on a 705 nt ssDNA substrate (Figure 4d). After 2 h of incubation, all ssDNA remained bound by RAD51 and there was no significant difference in the migration of nucleoprotein species with or without RAD54. Taken together with the observation that ssDNA without secondary structure does not support ATP hydrolysis by RAD54 (18), these data demonstrate that RAD54 removes RAD51 specifically from dsDNA and not ssDNA.
Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.