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.
Highlights
The strand exchange protein RAD51 functions to promote genome stability by repairing DNA double strand breaks (DSB) and damaged replication forks [1,2,3]
Following nucleoprotein filament formation, RAD51 carries out a search for homologous dsDNA sequences and promotes invasion of target duplex leading to the exchange of DNA strands that forms heteroduplex DNA within an intermediate called the displacement loop (D-loop)
DNase treatment did not alter the staining pattern of the nuclear matrix protein Lamin B, a protein that does not associate with DNA (Supplementary Figure S2a) [40,41]. These results indicate that RAD51 fibers that form as a consequence of overexpression represent a DNA bound form of the protein
Summary
The strand exchange protein RAD51 functions to promote genome stability by repairing DNA double strand breaks (DSB) and damaged replication forks [1,2,3]. RAD51 repairs damage by forming helical nucleoprotein filaments on tracts of ssDNA. Such tracts form by 5 -3 processing of DNA ends formed by DSBs, and as a consequence of replication fork collapse or blockage. Following nucleoprotein filament formation, RAD51 carries out a search for homologous dsDNA sequences and promotes invasion of target duplex leading to the exchange of DNA strands that forms heteroduplex DNA within an intermediate called the displacement loop (D-loop). The ssDNA strand displaced from the target duplex during heteroduplex DNA formation binds RPA [5]. RAD51 focus formation can be induced by treatments that damage DNA or inhibit replication, and the majority of these damage-induced RAD51 foci co-localize with RPA
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