The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons
I have established a method of expressing and purifying a 1,127 amino acid fragment of human BRCA2 (BRCA2<sub>BRC1-8</sub>) in which all eight BRC repeats are housed, providing a novel tool for research into mammalian recombination. Structural and functional studies of BRCA<sub>BRC...
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ndltd-bl.uk-oai-ethos.bl.uk-5983482015-03-20T06:05:14ZThe interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exonsDavies, O. R.2007I have established a method of expressing and purifying a 1,127 amino acid fragment of human BRCA2 (BRCA2<sub>BRC1-8</sub>) in which all eight BRC repeats are housed, providing a novel tool for research into mammalian recombination. Structural and functional studies of BRCA<sub>BRC1-8 </sub>have provided important insights into the nature of the BRC repeat-containing region of BRCA2 as a whole. Furthermore, through collaborative research, BRCA2<sub>BRC1-8 </sub>was found to promote RAD51-mediated strand exchange <i>in vitro. </i>This establishes a collective function for the multiple BRC repeat region of BRCA2, providing the first evidence that the BRC repeats of BRCA2 function as recombination mediators. An additional unrelated RAD51-binding site of currently unknown function is located at the C-terminal exon 27-encoded region of BRCA2 (BRCA2<sub>Exon27</sub>). I have established that, in contrast to the inhibitory action of BRC repeats, BRCA2<sub>Exon27 </sub>binds RAD51 through a non-inhibitory mechanism and further associates with RAD51-DNA nucleoprotein filaments. Furthermore, I have demonstrated a function of BRCA2<sub>Exon27</sub> in protecting RAD51-ssDNA nucleoprotein filaments from disruption by BRC repeats. This function of BRCA2<sub>Exon27 </sub>is not extended to oligomeric RAD51 in isolation or RAD51-dsDNA complexes, highlighting that BRCA2<sub>Exon27 </sub>specifically protects RAD51-ssDNA nucleoprotein filaments, the key structures required for recombination-mediated repair. These findings suggest a model in which active recombination requires an interplay between the disruptive role of BRC repeats and the protective role of BRCA2<sub>Exon27</sub>. Within the context of BRCA2<sub>BRC1-8</sub>, the disruptive function of BRC repeats may load RAD51 onto ssDNA to form the nucleoprotein filament. This filament is then protected from subsequent BRC repeat-dependent disruption by BRCA2<sub>Exon27 </sub>for the duration of active recombination. It has previously been reported that BRCA2<sub>Exon27</sub> is phosphorylated at the G<sub>2</sub>-M phase cell cycle transition, and upon phosphorylation can no longer interact with RAD51. I have demonstrated that the protection function of BRCA2<sub>Exon27 </sub>is abrogated by its phosphorylation. This further suggests a model for the known termination of recombination during mitosis; once phosphorylated, the protective function of BRCA2<sub>Exon27</sub> is lost and the disruptive function of BRC repeats may then dominate in disrupting all remaining RAD51-DNA nucleoprotein filaments, thus terminating recombination.616.994University of Cambridgehttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598348Electronic Thesis or Dissertation |
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616.994 Davies, O. R. The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
description |
I have established a method of expressing and purifying a 1,127 amino acid fragment of human BRCA2 (BRCA2<sub>BRC1-8</sub>) in which all eight BRC repeats are housed, providing a novel tool for research into mammalian recombination. Structural and functional studies of BRCA<sub>BRC1-8 </sub>have provided important insights into the nature of the BRC repeat-containing region of BRCA2 as a whole. Furthermore, through collaborative research, BRCA2<sub>BRC1-8 </sub>was found to promote RAD51-mediated strand exchange <i>in vitro. </i>This establishes a collective function for the multiple BRC repeat region of BRCA2, providing the first evidence that the BRC repeats of BRCA2 function as recombination mediators. An additional unrelated RAD51-binding site of currently unknown function is located at the C-terminal exon 27-encoded region of BRCA2 (BRCA2<sub>Exon27</sub>). I have established that, in contrast to the inhibitory action of BRC repeats, BRCA2<sub>Exon27 </sub>binds RAD51 through a non-inhibitory mechanism and further associates with RAD51-DNA nucleoprotein filaments. Furthermore, I have demonstrated a function of BRCA2<sub>Exon27</sub> in protecting RAD51-ssDNA nucleoprotein filaments from disruption by BRC repeats. This function of BRCA2<sub>Exon27 </sub>is not extended to oligomeric RAD51 in isolation or RAD51-dsDNA complexes, highlighting that BRCA2<sub>Exon27 </sub>specifically protects RAD51-ssDNA nucleoprotein filaments, the key structures required for recombination-mediated repair. These findings suggest a model in which active recombination requires an interplay between the disruptive role of BRC repeats and the protective role of BRCA2<sub>Exon27</sub>. Within the context of BRCA2<sub>BRC1-8</sub>, the disruptive function of BRC repeats may load RAD51 onto ssDNA to form the nucleoprotein filament. This filament is then protected from subsequent BRC repeat-dependent disruption by BRCA2<sub>Exon27 </sub>for the duration of active recombination. It has previously been reported that BRCA2<sub>Exon27</sub> is phosphorylated at the G<sub>2</sub>-M phase cell cycle transition, and upon phosphorylation can no longer interact with RAD51. I have demonstrated that the protection function of BRCA2<sub>Exon27 </sub>is abrogated by its phosphorylation. This further suggests a model for the known termination of recombination during mitosis; once phosphorylated, the protective function of BRCA2<sub>Exon27</sub> is lost and the disruptive function of BRC repeats may then dominate in disrupting all remaining RAD51-DNA nucleoprotein filaments, thus terminating recombination. |
author |
Davies, O. R. |
author_facet |
Davies, O. R. |
author_sort |
Davies, O. R. |
title |
The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
title_short |
The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
title_full |
The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
title_fullStr |
The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
title_full_unstemmed |
The interplay between breast cancer susceptibility protein BRCA2 and RAD51 in homologous recombination : a tale of two exons |
title_sort |
interplay between breast cancer susceptibility protein brca2 and rad51 in homologous recombination : a tale of two exons |
publisher |
University of Cambridge |
publishDate |
2007 |
url |
http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598348 |
work_keys_str_mv |
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