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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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Related in: MedlinePlus

Homologous recombination (HR) between direct and inverted repeats. HR reconstitutes a functional PuroR 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 PuroR 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 HygR gene between inverted Puro repeats (a) or deletes HygR between direct Puro repeats (b). Deletion events can also result from SCRS or SSA (see text)
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Fig1: Homologous recombination (HR) between direct and inverted repeats. HR reconstitutes a functional PuroR 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 PuroR 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 HygR gene between inverted Puro repeats (a) or deletes HygR between direct Puro repeats (b). Deletion events can also result from SCRS or SSA (see text)

Mentions: The MCF-7 cell lines employed as backgrounds for EMSY overexpression each carries a single, intact copy of a recombination/repair construct containing either a direct-repeat or an inverted-repeat of two inactive PuroR genes separated by the HygR gene (Fig. 1; Lemelin et al. 2005). Deleting EagI and BssHII restriction sites and inserting one I-SceI cleavage site inactivated the full-length Puro gene, whereas the wt Puro gene contained an inactivating 5′ deletion, including the promoter. A recombination/repair event between the two Puro cassettes would reconstitute a functional PuroR gene through loss of I-SceI and gain of wt EagI/BssHII sites, restoring resistance to the drug puromycin in colony assay.Fig. 1


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)

Homologous recombination (HR) between direct and inverted repeats. HR reconstitutes a functional PuroR 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 PuroR 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 HygR gene between inverted Puro repeats (a) or deletes HygR between direct Puro repeats (b). Deletion events can also result from SCRS or SSA (see text)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3064890&req=5

Fig1: Homologous recombination (HR) between direct and inverted repeats. HR reconstitutes a functional PuroR 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 PuroR 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 HygR gene between inverted Puro repeats (a) or deletes HygR between direct Puro repeats (b). Deletion events can also result from SCRS or SSA (see text)
Mentions: The MCF-7 cell lines employed as backgrounds for EMSY overexpression each carries a single, intact copy of a recombination/repair construct containing either a direct-repeat or an inverted-repeat of two inactive PuroR genes separated by the HygR gene (Fig. 1; Lemelin et al. 2005). Deleting EagI and BssHII restriction sites and inserting one I-SceI cleavage site inactivated the full-length Puro gene, whereas the wt Puro gene contained an inactivating 5′ deletion, including the promoter. A recombination/repair event between the two Puro cassettes would reconstitute a functional PuroR gene through loss of I-SceI and gain of wt EagI/BssHII sites, restoring resistance to the drug puromycin in colony assay.Fig. 1

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