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

Abstract

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.

The RAD51-Stimulatory Compound RS-1 Can Exploit the RAD51 Overexpression That Exists in Cancer Cells and Tumors

“…Cellular resistance to RAD51 stimulation depends on RAD54B and RAD54L protein levels, consistent with the ability of Swi2/Snf2-related translocases to remove aberrant RAD51 complexes from undamaged chromatin(18, 20). We found, however, that RAD54B depletion results in greater RS-1 sensitization than RAD54L depletion.…”

“…This cytologic observation coincides with slower cell growth and elevated genomic instability(18). Translocase depletion can also result in accumulation of non-damage-associated RAD51 complexes bound to DNA in human tumor cells(20). Therefore, the propensity for cancer cells to form toxic RAD51 complexes likely reflects an imbalance between RAD51 protein concentration and the combined activities of RAD54 family translocases.…”

“…Taken together, RAD51 overexpression may be a common mechanism leading to genomic instability, which in turn fuels malignant progression of human cancers. Analysis of tumor cells containing high levels of non-damage-associated RAD51 complexes indicates that defects in chromosome segregation underlie this instability (20). …”

Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation

“…We found that rad54Δ increases the loss rate to a greater extent than rad51Δ (Figure 1C). Rad54-specific functions such as chromatin remodeling (48–50), rather than the ineffective attempts of recombination by Rad51 in rad54Δ cells (51), may account for the high rate of chromosome loss, as rad51Δ rad54Δ increased the loss rate to the same level as rad54Δ . Histone H3K9 methyltransferase Clr4 is required for the heterochromatin structure in centromeres (52,53), and clr4Δ cells are hypersensitive to a microtubule-destabilizing drug, thiabendazole (TBZ) (54).…”

DNA repair targeted therapy: The past or future of cancer treatment?

“…RS-1 acts as an allosteric effector of active hRad51 filament formation on ssDNA that stimulates DNA binding and recombination activities of hRad51 by locking its active conformation without affecting active site ATP hydrolysis. RS-1 has so far been demonstrated to have single agent activity in tumor cell lines that display increased Rad51 expression and its activity is dependent on Rad51 and the Rad54B/Rad54L translocase (Mason et al, 2015). In addition, RS-1 has in vivo anti-cancer activity in a xenograft animal model (Mason et al, 2014).…”