Short EMSY expression in MCF-7 cell lines. PCR of genomic DNA was performed to demonstrate EMSY transgene integration. The primer pair employed amplifies only the EMSY transgene. Its mRNA was analyzed by qualitative RT-PCR with the same primer pair. As a control, a fragment from endogenous full-length EMSY was amplified in the same reaction. 18S rRNA was amplified as a control for RT-PCR. Water or no reverse transcriptase served as a negative control, whereas EMSY-expressing vector DNA was used as a positive control. EMSY proteins were assessed by Western blotting. All the different EMSY cell lines examined are independent cell clones

A model for the BRCA2/RAD51 pathway in response to DSBs. Not all proteins and protein–protein interactions are shown and the outlines are for illustration only. a After their recruitment by BRCA1/MRN, BRCA2/RAD51 complexes act upstream and downstream of ATR (see text for details). b After disruption of nucleosomes and transformation of DSB ends into ssDNA by BRCA1/MRN and ATM (1), RPA/BRCA2/RAD51 recruits ATR (2) to enhance SCC/alignment before the formation/stabilization of D-loop at which the 3′-ssDNA end primes new DNA synthesis (3). ATR recruitment/activation could also occur during or after strand invasion and D-loop formation/stabilization, since transformation of DSB ends into 3′-ssDNA could be performed by the unwinding activity of MRN rather than its exonuclease activity that normally yields 5′-ssDNA ends. In this case, unwinding, strand invasion and D-loop formation/stabilization could occur simultaneously after the search for homology, alignment/pairing between DSB ends and the intact homologous dsDNA partner. In either case, the resulting intermediate can either disassemble, the newly synthesized strand can be displaced by unwinding to anneal with the non-invading 3′-ssDNA end to elicit non-crossover gene conversion only, or be processed to yield gene conversion with or without crossover (4). Short EMSY interferes with ATR recruitment/activation by overriding RPA (5), without affecting the ability of BRCA2/RAD51 to bind ssDNA and perform SSA (6). At D-loops, such as at gene promoters and replication origins, free RAD51 oligomers could also compete with RNA and DNA polymerases for ssDNA and thereby block initiation of transcription, replication and repair until activation/recruitment of PIKKs that would disrupt RAD51 oligomers into monomers. The loading of RAD51 oligomers by BRCA2_ex27 on ssDNA may also act similarly until disruption of RAD51 filament by post-translational modifications (i.e., phosphorylation, ubiquitylation, sumoylation) that would allow transcription, replication and repair to proceed and thereby resume cell-cycle arrest. In this case, transcription and replication factors could act as cellular GPS (guiding position system) elements, recruiting checkpoint proteins to specific sites. Such BRCA2/RAD51 transcription/replication-checkpoint function could explain why checkpoint-deficient cells exhibit radio-resistant DNA synthesis, a characteristic feature of CIN syndromes, including BRCA-deficiency (see text). Thus, whereas RAD51 oligomer can act as a transcription/replication/repair repressor upstream of PIKKs, BRCA2/RAD51 complex can behave as both an activator and a repressor upstream and downstream of PIKKs, respectively

Homologous recombination (HR) between direct and inverted repeats. HR reconstitutes a functional Puro^R gene through loss of the I-SceI site and gain of EagI/BssHII sites. Since I-SceI insertion mutation in ScePuro entails the deletion of EagI and BssHII sites, only error-free HR repair events between the two Puro^R cassettes can restore a functional Puro gene. Gene conversion restores one functional Puro gene without affecting the overall structure of the locus, whereas crossover associated or not with gene conversion inverts Hyg^R gene between inverted Puro repeats (a) or deletes Hyg^R between direct Puro repeats (b). Deletion events can also result from SCRS or SSA (see text)

Effect of short EMSY on BRCA2 and RAD51 expression. RT-PCR reveals no apparent effect of short EMSY on RAD51 or BRCA2 expression levels. 18S rRNA was amplified as a control for RT-PCR. Water or no reverse transcriptase was a negative control. Western blotting analysis indicates no apparent effect of short EMSY on RAD51 protein level. Actin served as loading control

A model for the BRCA2/RAD51 pathway in the response to stalled forks. After their recruitment by BRCA1/MRN, BRCA2/RAD51 complexes may act in concert to (i) disassemble chromatin (A); (ii) maintain SCC/alignment (B); (iii) effectuate strand-switching and thereby recruit/activate ATR to enhance SCC/alignment (C); (iv) provide one DSB end, and thereby activate ATM and DNA-PK (D); and (v) twist the DSB end after its processing at least by error-free TLS polymerases into leading and lagging strands that would anneal back with the original templates, and thereby yield a hemicatenane (E). If needed, the harmless DSB end (D and F) may engage in HR, NHEJ or SSA between neighbouring stalled forks during termination of the replication (S) phase (not shown). By interfering with ATR recruitment/activation, short EMSY could induce RFR (RAD51-independent HR) and chicken-foot formation (F). HJ-resolving enzymes against crossover, such as BLM and WRN, may prevent fork collapse (G) by competing with HJ-nicking enzymes (red arrows) at the expense of CIN. Unlike hyper-recombination, BRCA2/RAD51-mediated strand switching at either stalled forks (hemicatenation/RFR) or DSBs does not require RAD51-filament formation, in which case the fourfold decrease in HR repair of I-SceI-induced DSBs by short EMSY could be attributed to apoptosis/senescence rather than decreased efficiency of strand switching. Consistently, meiotic HR does not require the loading of RAD51 oligomers by BRCA2 at programmed DSBs, since BRCA2^∆ex27 KO mice are as fertile as wild-type animals, except that they exhibit hypersensitivity to IR, senescence and reduced life span. Thus, whereas BRCA2/RAD51-mediated strand switching ensures survival, maintains chromosomal stability and acts as an anti-cancer barrier, SCRS and RAD51-mediated strand exchange/invasion can compete with RFR for ssDNA and thereby decrease the likelihood of forks collapse at the expense of CIN and human diseases. Only abnormally proliferating cells crippled in an essential gene are prone to gene mutation/reversion and fork collapse