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

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. 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. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. 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.

Competitive PCR. We employed competitive PCR to determine the amount of depletion of mRNA. RNA was harvested from cells using a High Pure RNA Isolation Kit (Roche). 0.5 µg of total RNA was reverse-transcribed using MLV-RT (Qiagen) with a dT (20) primer at 42 o C for 1 hour. To quantitate mRNA levels, competitive PCR was performed as previously described (4). Briefly, competitor fragments for RAD54L and RAD54B were generated by inserting a 20 bp sequence (5' GTCGACGGATCCCTGCAGGT) into the product amplified by gene-specific PCR primers. The relative amount of RNA was determined by co-amplification of cDNA with increasing amounts of competitor fragments. Products were run on 2 % agarose gels in 1X TBE and band intensity was measured using ImageJ. The ratio of band intensity of Competitor (C) to target cDNA (T) was determined and plotted. The amount of cDNA amplified in each reaction was determined by identifying the amount of competitor that resulted in a C/T ratio of 1. For all RT reactions, GAPDH was used as a loading control.
Determination of the concentration of RAD51 in the nucleus. MCF7 cells were harvested, counted, and lysed as above. Whole cells extract were loaded on an SDS-PAGE gel along with increasing amounts of human RAD51 protein. Protein was transferred to PVDF and probed with RAD51 antibody. Band intensity was quantitated using ImageJ. To calculate the concentration of RAD51 in tumor cells, the mass of RAD51 protein per lane was determined using a standard curve generated with purified RAD51 protein. The mass of RAD51/cells was then determined by dividing the mass per lane by the number of cell equivalents loaded.
RAD51 concentration was then calculated using a nuclear volume of 1x10 -12 . This volume was calculated based on measurement of the average nuclear diameter (12.4 µm). We also assumed that 70% of total RAD51 is localized in the nucleus (5). Proteins. Purification of yeast Rad51 (7), yeast Rad54 (8), human RAD54 (9), human RAD51(10) were performed as described. and the incubation continued for two minutes. At this time, 30 % of the reaction (5.7 µl) was removed and fixed with 0.25 % glutaraldehyde and immediately afterward 300 µM bp of Xmn1-linearized pUC19 DNA was added (0.7 µl of 600 µM) to the remainder of the reaction and incubation continued. At one and two hours past addition of pUC19 scavenger, 30 % of the reaction (6 µl) was removed and fixed with glutaraldehyde. About half an hour after fixation of the last time point, DNA loading dye was added to the samples, which were then loaded on a 1 % agarose TAE gel and electrophoresed for 100 minutes at 4 V/cm. Gels were then vacuum-dried on DE-81 (Whatman) paper and exposed to a storage phosphor screen overnight and imaged on a Storm 860 scanner. Reactions were quantitated using ImageQuant software by normalizing the freely migrating DNA species (non-shifted mobility) to an equivalent mass of 32 P DNA electrophoresed without protein as equal to 100 % free substrate. DNA Substrates: Linear dsDNA substrate (~3 kb) was generated by PCR amplification of pBluescript plasmid DNA, followed by ethanol precipitation. The DNA in the pellet was dissolved and passed through a PCR Kleen (BioRad) spin column to remove primers and excess dNTPs. The resulting DNA (about 10 µg) was 5' end labeled with 30 U of T4 polynucleotide kinase and 50 µCi γ32 P-ATP in a volume of 200 µl.

Antibodies. RAD51 antibody was a kind gift from
After 1 hour incubation at 37 °C, the reactions were purified with a PCR purification kit (Bioneer) and quantified with a Nanodrop spectrophotometer. 705 nt ssDNA was produced from phagemid circular ssDNA as described (11). This DNA was 5' end labeled and re-purified as described above for the dsDNA substrate.
XmnI-linearized pUC19 DNA 'scavenger DNA' was produced by plasmid digestion followed by two rounds of phenol/chloroform extraction, chloroform extraction and ethanol precipitation. Figure 1. Analysis of expression levels. (a) The concentration of RAD51 in tumor cells was estimated by quantitative western using decreasing amounts of purified human RAD51 protein as a standard curve. Whole cells extracts were loaded onto the western blot along with the indicated amount of purified human RAD51. The estimated cellular concentration of RAD51 is noted below the blot. α-Tubulin was used as a loading control. (b,c) Western blots depicting RAD54L and RAD54B protein levels after transfection with the indicated siRNAs. (d) Competitive PCR to determine mRNA levels after indicated siRNA transfections. PCR was carried out with increasing concentration of competitor (see Supplemental Material and Methods for details). Representative graph plotting the ratio of Rad54l band intensities between competitor (C) and target cDNA (T) in cells transfected with NS or RAD54L+B siRNAs. The amount of competitor used in the PCR reaction is depicted on the x-axis (e) Graph depicting the level of Rad54l mRNA after transfection with RAD54L+B siRNAs compared to the NS control. Graph depicts average of two independent reactions. (f) Representative graphs measuring Rad54b levels as described in c (g) Graph depicting the level of Rad54b mRNA after transfection with RAD54L+B siRNAs compared to the NS control. Graph depicts average of two independent reactions. Error bars are standard error.