Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

The dichotomous effects of caffeine on homologous recombination in mammalian cells.

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.

Nascent DNA synthesis during homologous recombination is synergistically promoted by the rad51 recombinase and DNA homology.

In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3' extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3' extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3' extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3' extension. Overexpression of wild-type Rad51 stimulated the frequency of 3' extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3' extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3' extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3' extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3' extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 3' extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3' extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization.

Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing.

Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.

High levels of wild-type BRCA2 suppress homologous recombination.

Endogenous levels of the BRCA2 (breast cancer susceptibility 2) protein promote homologous recombination by regulating the essential strand exchange protein RAD51. To examine BRCA2 function in homologous recombination, we expressed human BRCA2 in control mouse hybridoma cells, as well as those that were depleted of endogenous Brca2 by small interfering RNA. With moderate human BRCA2 expression, homologous recombination was stimulated. Conversely, a higher level of BRCA2 reduced homologous recombination and DNA-damage-induced Rad51 foci formation. Cells expressing high levels of BRCA2 feature normal growth, increased sensitivity to mitomycin C, and increased illegitimate recombination. BRCA2-overexpressing cells are also characterized by suppression of p53 transcriptional regulation and a corresponding reduction in the expression of the p53-responsive genes Noxa and p21. Notably, in cells expressing high levels of BRCA2, small interfering RNA depletion of human BRCA2 or ectopic expression of Rad51 increases homologous recombination and decreases illegitimate recombination. Thus, high levels of wild-type BRCA2 perturb Rad51-mediated homologous recombination, and relatively normal recombination responses can be restored by rebalancing recombination factors.

A strand invasion 3' polymerization intermediate of mammalian homologous recombination.

Initial events in double-strand break repair by homologous recombination in vivo involve homology searching, 3' strand invasion, and new DNA synthesis. While studies in yeast have contributed much to our knowledge of these processes, in comparison, little is known of the early events in the integrated mammalian system. In this study, a sensitive PCR procedure was developed to detect the new DNA synthesis that accompanies mammalian homologous recombination. The test system exploits a well-characterized gene targeting assay in which the transfected vector bears a gap in the region of homology to the single-copy chromosomal immunoglobulin mu heavy chain gene in mouse hybridoma cells. New DNA synthesis primed by invading 3' vector ends copies chromosomal mu-gene template sequences excluded by the vector-borne double-stranded gap. Following electroporation, specific 3' extension products from each vector end are detected with rapid kinetics: they appear after 0.5 hr, peak at 3-6 hr, and then decline, likely as a result of the combined effects of susceptibility to degradation and cell division. New DNA synthesis from each vector 3' end extends at least approximately 1000 nucleotides into the gapped region, but the efficiency declines markedly within the first approximately 200 nucleotides. Over this short distance, an average frequency of 3' extension for the two invading vector ends is approximately 0.007 events/vector backbone. DNA sequencing reveals precise copying of the cognate chromosomal mu-gene template. In unsynchronized cells, 3' extension is sensitive to aphidicolin supporting involvement of a replicative polymerase. Analysis suggests that the vast majority of 3' extensions reside on linear plasmid molecules.