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RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

Mason JM, Dusad K, Wright WD, Grubb J, Budke B, Heyer WD, Connell PP, Weichselbaum RR, Bishop DK - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Cummings Life Science Center, Box 13, 920 East 58th St., Chicago, IL 60637, USA.

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Human RAD54 disassembles human RAD51-dsDNA filaments in vitro. (a) Scheme of electrophoretic mobility shift assay (EMSA) for RAD54-mediated disassembly of RAD51-dsDNA filaments. A 3 kilobase dsDNA substrate (6 μM bp) that is 5′ end-labeled with 32P is incubated with RAD51 (1.5 μM) for 20 min at room temperature (∼23°C). RAD54 (100 nM) is then added, or identical buffer, and incubation continued for 2 min. A 30 μM bp pUC19 ‘scavenger’ dsDNA is then added to bind RAD51 protein that is removed by RAD54, preventing rebinding to the original dsDNA. After 1 or 2 h, the reaction is stopped by fixation with 0.25% glutaraldehyde and subject to TAE-agarose gel electrophoresis. (b) Phosphorimage of EMSA performed with human or yeast Rad51+/- Rad54 cognate pairs. Reactions contained 4 mM MgCl2 and CaCl2 where indicated. (c) Calcium titration into the disassembly reaction, with 4 mM magnesium ion and the indicated concentration of calcium. Quantitation corresponding to each lane condition is provided underneath the gel image. The first lane (marked ‘s’) contains DNA substrate and no protein and represents 100% signal to which the other lanes’ free DNA are normalized. Error bars are the standard deviation of three independent experiments. (d) Experiment performed with a 705 nt ssDNA substrate (6 μM nucleotide) and 0.5 mM CaCl2. The doublet band seen in the substrate only lane, marked ‘s’, is likely due to alternative secondary structural forms of the ssDNA substrate. Error bars are the standard deviation of three independent experiments.
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Figure 4: Human RAD54 disassembles human RAD51-dsDNA filaments in vitro. (a) Scheme of electrophoretic mobility shift assay (EMSA) for RAD54-mediated disassembly of RAD51-dsDNA filaments. A 3 kilobase dsDNA substrate (6 μM bp) that is 5′ end-labeled with 32P is incubated with RAD51 (1.5 μM) for 20 min at room temperature (∼23°C). RAD54 (100 nM) is then added, or identical buffer, and incubation continued for 2 min. A 30 μM bp pUC19 ‘scavenger’ dsDNA is then added to bind RAD51 protein that is removed by RAD54, preventing rebinding to the original dsDNA. After 1 or 2 h, the reaction is stopped by fixation with 0.25% glutaraldehyde and subject to TAE-agarose gel electrophoresis. (b) Phosphorimage of EMSA performed with human or yeast Rad51+/- Rad54 cognate pairs. Reactions contained 4 mM MgCl2 and CaCl2 where indicated. (c) Calcium titration into the disassembly reaction, with 4 mM magnesium ion and the indicated concentration of calcium. Quantitation corresponding to each lane condition is provided underneath the gel image. The first lane (marked ‘s’) contains DNA substrate and no protein and represents 100% signal to which the other lanes’ free DNA are normalized. Error bars are the standard deviation of three independent experiments. (d) Experiment performed with a 705 nt ssDNA substrate (6 μM nucleotide) and 0.5 mM CaCl2. The doublet band seen in the substrate only lane, marked ‘s’, is likely due to alternative secondary structural forms of the ssDNA substrate. Error bars are the standard deviation of three independent experiments.

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.


RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

Mason JM, Dusad K, Wright WD, Grubb J, Budke B, Heyer WD, Connell PP, Weichselbaum RR, Bishop DK - Nucleic Acids Res. (2015)

Human RAD54 disassembles human RAD51-dsDNA filaments in vitro. (a) Scheme of electrophoretic mobility shift assay (EMSA) for RAD54-mediated disassembly of RAD51-dsDNA filaments. A 3 kilobase dsDNA substrate (6 μM bp) that is 5′ end-labeled with 32P is incubated with RAD51 (1.5 μM) for 20 min at room temperature (∼23°C). RAD54 (100 nM) is then added, or identical buffer, and incubation continued for 2 min. A 30 μM bp pUC19 ‘scavenger’ dsDNA is then added to bind RAD51 protein that is removed by RAD54, preventing rebinding to the original dsDNA. After 1 or 2 h, the reaction is stopped by fixation with 0.25% glutaraldehyde and subject to TAE-agarose gel electrophoresis. (b) Phosphorimage of EMSA performed with human or yeast Rad51+/- Rad54 cognate pairs. Reactions contained 4 mM MgCl2 and CaCl2 where indicated. (c) Calcium titration into the disassembly reaction, with 4 mM magnesium ion and the indicated concentration of calcium. Quantitation corresponding to each lane condition is provided underneath the gel image. The first lane (marked ‘s’) contains DNA substrate and no protein and represents 100% signal to which the other lanes’ free DNA are normalized. Error bars are the standard deviation of three independent experiments. (d) Experiment performed with a 705 nt ssDNA substrate (6 μM nucleotide) and 0.5 mM CaCl2. The doublet band seen in the substrate only lane, marked ‘s’, is likely due to alternative secondary structural forms of the ssDNA substrate. Error bars are the standard deviation of three independent experiments.
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Figure 4: Human RAD54 disassembles human RAD51-dsDNA filaments in vitro. (a) Scheme of electrophoretic mobility shift assay (EMSA) for RAD54-mediated disassembly of RAD51-dsDNA filaments. A 3 kilobase dsDNA substrate (6 μM bp) that is 5′ end-labeled with 32P is incubated with RAD51 (1.5 μM) for 20 min at room temperature (∼23°C). RAD54 (100 nM) is then added, or identical buffer, and incubation continued for 2 min. A 30 μM bp pUC19 ‘scavenger’ dsDNA is then added to bind RAD51 protein that is removed by RAD54, preventing rebinding to the original dsDNA. After 1 or 2 h, the reaction is stopped by fixation with 0.25% glutaraldehyde and subject to TAE-agarose gel electrophoresis. (b) Phosphorimage of EMSA performed with human or yeast Rad51+/- Rad54 cognate pairs. Reactions contained 4 mM MgCl2 and CaCl2 where indicated. (c) Calcium titration into the disassembly reaction, with 4 mM magnesium ion and the indicated concentration of calcium. Quantitation corresponding to each lane condition is provided underneath the gel image. The first lane (marked ‘s’) contains DNA substrate and no protein and represents 100% signal to which the other lanes’ free DNA are normalized. Error bars are the standard deviation of three independent experiments. (d) Experiment performed with a 705 nt ssDNA substrate (6 μM nucleotide) and 0.5 mM CaCl2. The doublet band seen in the substrate only lane, marked ‘s’, is likely due to alternative secondary structural forms of the ssDNA substrate. Error bars are the standard deviation of three independent experiments.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus