Our results reveal the biochemical functions of full length human BRCA2, and they establish that BRCA2 augments the functions of RAD51 that are essential for recombinational repair of DNA breaks (Supplementary Fig. 1
). Stimulation by BRCA2 is a consequence of several mutually reinforcing effects; BRCA2: 1) enforces binding of RAD51 to ssDNA; 2) accelerates the rate of RPA-displacement from ssDNA by RAD51; 3) inhibits the ATPase activity of RAD51; and 4) limits binding to dsDNA. BRCA2 focuses the assembly of RAD51 onto ssDNA and facilitates the RAD51-mediated displacement of RPA from ssDNA, which is a key regulatory step of DNA pairing. In support of this general concept, the promotion of RAD51 filament formation onto RPA-coated ssDNA was also demonstrated by Liu et al.
(submitted) using a different full-length BRCA2 protein expression construct and preparation. By enabling formation of the presynaptic complex, BRCA2 permits progression to the subsequent DNA pairing phase of recombinational DNA repair. Furthermore, by inhibiting the ssDNA-dependent ATP hydrolysis of RAD51, BRCA2 preserves the active and most stable form of RAD51, the ATP-RAD51-ssDNA complex21,22
. Because the rate-limiting step in RAD51 nucleoprotein filament assembly is nucleation of the first several monomers of the filament45–47
, BRCA2 can act catalytically to stabilize a nucleus by blocking RAD51 self-inactivation and dissociation via
its ATPase activity. If the RAD51 molecules bound to BRCA2 do indeed comprise the nucleus, then BRCA2 can stabilize a nascent filament of up to 4–6 RAD51 molecules. Inhibition of ATPase activity was also observed for the C. elegans
proteins (RAD51 and BRC-2)23
, but not for the U. maydis
orthologues (Rad51 and Brh2)17
or the E. coli
analogues (RecA and RecFOR)36
, suggesting that this mechanism of stimulating RAD51 function is a late adaption of multicellular organisms. In addition, our results show that BRCA2 prevents or slows the assembly of RAD51 onto duplex DNA, an aspect of RAD51 filament assembly that impairs recombination reactions. Based on previous single-molecule studies with the BRC repeats21
, we propose that interaction with full-length BRCA2 slows nucleation of RAD51 onto dsDNA.
An unanticipated feature of human BRCA2 is its ability to bind and stimulate RAD51-mediated DNA strand exchange at regions of ssDNA as well as at ssDNA/dsDNA junctions of either polarity. This capability permits the BRCA2-facilitated loading of RAD51 in both the 5’→3’ and 3’→5’ directions. Although most models for BRCA2 function have focused on DSB repair, we note that these characteristics are also consistent and suggestive of a role for BRCA2 in the recombinational repair of DNA gaps that occur during DNA replication due to damage in the template (Supplementary Fig. 1b
). Given that BRCA2 facilitates growth of the filament in either polarity from internal ssDNA regions or from either junction, BRCA2 could readily contribute to daughter strand DNA gap repair as well as DSB repair. It is notable that the lack of polarity in human BRCA2 function differs from analogs such as RecFOR and Brh2, which load RecA and U. maydis
Rad51, respectively, specifically onto the 3’ overhanging ssDNA17,36
. The absence of a bias for pairing 3’-ends by human RAD51 may reflect its underlying intrinsic capability to assemble in both directions48
. But because resection of a DSB in vivo
produces 3’ tailed ssDNA, there is no compelling need for polarity enforcement by BRCA2.
Another key difference between the BRCA2 orthologues of U. maydis
and C. elegans
is that human BRCA2 is unable to anneal ssDNA complexed with RPA, the physiological intermediate of recombination. However, human RAD52 does manifest this capability, implying that in mammalian cells this function is assumed by RAD52. Yet RAD52 cannot stimulate the assembly of RAD51 onto ssDNA complexed with RPA40
(Supplementary Fig. 12
), but BRCA2 can. This behavior of RAD52 is distinct from yRad52 which displays both of these species-specific capabilities37–39,41
, explaining the essential role of yRad52 in all recombinational DNA repair processes. Thus, in vertebrates, these functions have separated: BRCA2 targets RAD51 to ssDNA to mediate DNA strand invasion into a duplex donor to produce joint molecules by a distinctive mechanism, whereas RAD52 anneals RPA-ssDNA complexes in steps or pathways of recombinational repair that could include second-end capture in DSB repair, single-strand annealing (SSA), and synthesis-dependent strand annealing. In support of this idea, error prone repair by SSA is increased in cells lacking functional BRCA2, whereas RAD52
complementation in RAD52−/−
mouse ES cells augments the SSA pathway44,49
. Both in vivo
and in vitro
studies clarified the role that BRCA2 plays in catalyzing the delivery of RAD51 to sites of DNA damage. Our work has confirmed and extended prior expectations that the intact human BRCA2 protein mediates the rapid and ordered assembly of the RAD51 protein onto ssDNA, and helps to explain why cells lacking functional BRCA2 would be severely impaired for formation of this critical intermediate in recombinational repair. As a consequence of BRCA2 loss, DNA break repair mediated through template-directed repair from homologous sequences within an intact homolog or sister chromosome would be disrupted, leading to error prone repair and potential chromosomal instability. The ability to now purify full length human BRCA2, a protein directly responsible for genetically predisposing individuals to substantially high risks for cancer, should open a whole new venue for understanding this very large and complex protein.