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EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases.

Cousineau I, Belmaaza A - Mol. Genet. Genomics (2011)

Bottom Line: EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer.Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression.Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.

ABSTRACT
EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.

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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 BRCA2ex27 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
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Fig4: 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 BRCA2ex27 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

Mentions: It has been shown that RAD51 phosphorylation cripples the ability of RAD51 to both bind ssDNA and undertake DNA strand-exchange/invasion in vitro and HR repair of I-SceI-induced chromosomal DSBs, presumably by disrupting RAD51 oligomers into monomers and preventing RAD51 oligomerization (Yuan et al. 1998; Daboussi et al. 2002; Conilleau et al. 2004). Thus, in response to DSBs, RAD51 phosphorylation would affect not only its function but also that of BRCA2, since de-phosphorylated BRCA2ex27 exclusively binds RAD51 oligomer but not RAD51 monomers (Thorslund and West 2007), raising the question: how does BRCA2/RAD51 complex conduct HR repair of DSBs? Since none of the models proposed for HR in general or for BRCA1/2 function in particular considers such a conundrum, we propose that BRCA2 may have the ability to protect its RAD51 monomers bound by the BRC repeats (exon 11) against phosphorylation (Abaji et al. 2005) (Fig. 4a).Fig. 4


EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases.

Cousineau I, Belmaaza A - Mol. Genet. Genomics (2011)

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 BRCA2ex27 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
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Fig4: 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 BRCA2ex27 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
Mentions: It has been shown that RAD51 phosphorylation cripples the ability of RAD51 to both bind ssDNA and undertake DNA strand-exchange/invasion in vitro and HR repair of I-SceI-induced chromosomal DSBs, presumably by disrupting RAD51 oligomers into monomers and preventing RAD51 oligomerization (Yuan et al. 1998; Daboussi et al. 2002; Conilleau et al. 2004). Thus, in response to DSBs, RAD51 phosphorylation would affect not only its function but also that of BRCA2, since de-phosphorylated BRCA2ex27 exclusively binds RAD51 oligomer but not RAD51 monomers (Thorslund and West 2007), raising the question: how does BRCA2/RAD51 complex conduct HR repair of DSBs? Since none of the models proposed for HR in general or for BRCA1/2 function in particular considers such a conundrum, we propose that BRCA2 may have the ability to protect its RAD51 monomers bound by the BRC repeats (exon 11) against phosphorylation (Abaji et al. 2005) (Fig. 4a).Fig. 4

Bottom Line: EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer.Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression.Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.

ABSTRACT
EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.

Show MeSH
Related in: MedlinePlus