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The carboxyl terminus of Brca2 links the disassembly of Rad51 complexes to mitotic entry.

Ayoub N, Rajendra E, Su X, Jeyasekharan AD, Mahen R, Venkitaraman AR - Curr. Biol. (2009)

Bottom Line: Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry.Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation.We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK.

ABSTRACT

Background: The Rad51 recombinase assembles on DNA to execute homologous DNA recombination (HR). This process is essential to repair replication-associated genomic lesions before cells enter mitosis, but how it is started and stopped during the cell cycle remains poorly understood. Rad51 assembly is regulated by the breast cancer suppressor Brca2, via its evolutionarily conserved BRC repeats, and a distinct carboxy (C)-terminal motif whose biological function is uncertain. Using "hit-and-run" gene targeting to insert single-codon substitutions into the avian Brca2 locus, we report here a previously unrecognized role for the C-terminal motif.

Results: We show that the avian C-terminal motif is functionally cognate with its human counterpart and identify point mutations that either abolish or enhance Rad51 binding. When these mutations are introduced into Brca2, we find that they affect neither the assembly of Rad51 into nuclear foci on damaged DNA nor DNA repair by HR. Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry. Conversely, the slower disassembly of foci in a point mutant that constitutively binds Rad51 correlates with delayed mitosis. Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation.

Conclusions: We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry. This mechanism may ensure that HR terminates before chromosome segregation. Our findings assign a biological function for the C-terminal Brca2 motif in a mechanism that coordinates DNA repair with the cell cycle.

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Normal Formation but Altered Dissolution of Damage-Induced Rad51 Foci in Brca2P3240L/− and Brca2T3232A/− Cells(A) Representation of a spot-counting algorithm on the Cellomics ArrayScan VTI. Left panel: a merged image of DNA (blue) and Rad51 (green). Middle and right panels: grayscale image of Rad51 exported to the Cellomics ArrayScan platform with (middle) and without (right) the algorithm overlay (details in Experimental Procedures).(B) Kinetics of Rad51 focus formation and dissolution in mimosine-synchronized Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells after exposure to 3 Gy ionizing radiation (IR). Five hundred cells were analyzed on the Cellomics ArrayScan for each data point, and the percentage of cells positive for Rad51 foci was plotted as a function of time (see Experimental Procedures for details). Nearly 100% of cells formed Rad51 foci after IR in all three cell lines by 3 hr after damage. However, decreased binding to Rad51 in the Brca2P3240L/− cell line correlated with a faster reduction in focus-positive cells compared to the heterozygous control. Conversely, the Brca2T3232A/− cell line showed a slower reduction in foci dissolution.
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fig4: Normal Formation but Altered Dissolution of Damage-Induced Rad51 Foci in Brca2P3240L/− and Brca2T3232A/− Cells(A) Representation of a spot-counting algorithm on the Cellomics ArrayScan VTI. Left panel: a merged image of DNA (blue) and Rad51 (green). Middle and right panels: grayscale image of Rad51 exported to the Cellomics ArrayScan platform with (middle) and without (right) the algorithm overlay (details in Experimental Procedures).(B) Kinetics of Rad51 focus formation and dissolution in mimosine-synchronized Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells after exposure to 3 Gy ionizing radiation (IR). Five hundred cells were analyzed on the Cellomics ArrayScan for each data point, and the percentage of cells positive for Rad51 foci was plotted as a function of time (see Experimental Procedures for details). Nearly 100% of cells formed Rad51 foci after IR in all three cell lines by 3 hr after damage. However, decreased binding to Rad51 in the Brca2P3240L/− cell line correlated with a faster reduction in focus-positive cells compared to the heterozygous control. Conversely, the Brca2T3232A/− cell line showed a slower reduction in foci dissolution.

Mentions: Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells synchronized in G1 and released into S phase were exposed to 3 Gy IR immediately after release and harvested every hour afterwards for analysis. Rad51 focus formation was enumerated in 500 cells for each data point by automated high-content microscopy (Figure 4A). The percentage of cells containing Rad51 foci was plotted as a function of time (Figure 4B). In all cell lines, nearly 100% of cells contained Rad51 foci by 3 hr after IR. However, in the Brca2P3240L/− cell line, there was a faster reduction in the percentage of focus-positive cells relative to wild-type, evident 4–5 hr after IR exposure. The P3240L mutation prevents Rad51 binding to the C-terminal Brca2 motif, suggesting that the loss of the interaction promotes this change. Consistent with this, accelerated dissipation of IR-induced Rad51 foci was also observed in Brca2S3239A/S3239A cells (Figure S6). This notion was further supported by the converse effect of the T3232A mutation, which enhanced Rad51 binding. In Brca2T3232A/− cells, there was a slower reduction in the percentage of focus-positive cells (Figure 4B). Together, these observations suggest that the C-terminal Rad51-binding motif in Brca2 regulates not the assembly of Rad51 foci at sites of DNA damage but rather the rate of their dissolution.


The carboxyl terminus of Brca2 links the disassembly of Rad51 complexes to mitotic entry.

Ayoub N, Rajendra E, Su X, Jeyasekharan AD, Mahen R, Venkitaraman AR - Curr. Biol. (2009)

Normal Formation but Altered Dissolution of Damage-Induced Rad51 Foci in Brca2P3240L/− and Brca2T3232A/− Cells(A) Representation of a spot-counting algorithm on the Cellomics ArrayScan VTI. Left panel: a merged image of DNA (blue) and Rad51 (green). Middle and right panels: grayscale image of Rad51 exported to the Cellomics ArrayScan platform with (middle) and without (right) the algorithm overlay (details in Experimental Procedures).(B) Kinetics of Rad51 focus formation and dissolution in mimosine-synchronized Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells after exposure to 3 Gy ionizing radiation (IR). Five hundred cells were analyzed on the Cellomics ArrayScan for each data point, and the percentage of cells positive for Rad51 foci was plotted as a function of time (see Experimental Procedures for details). Nearly 100% of cells formed Rad51 foci after IR in all three cell lines by 3 hr after damage. However, decreased binding to Rad51 in the Brca2P3240L/− cell line correlated with a faster reduction in focus-positive cells compared to the heterozygous control. Conversely, the Brca2T3232A/− cell line showed a slower reduction in foci dissolution.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2719694&req=5

fig4: Normal Formation but Altered Dissolution of Damage-Induced Rad51 Foci in Brca2P3240L/− and Brca2T3232A/− Cells(A) Representation of a spot-counting algorithm on the Cellomics ArrayScan VTI. Left panel: a merged image of DNA (blue) and Rad51 (green). Middle and right panels: grayscale image of Rad51 exported to the Cellomics ArrayScan platform with (middle) and without (right) the algorithm overlay (details in Experimental Procedures).(B) Kinetics of Rad51 focus formation and dissolution in mimosine-synchronized Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells after exposure to 3 Gy ionizing radiation (IR). Five hundred cells were analyzed on the Cellomics ArrayScan for each data point, and the percentage of cells positive for Rad51 foci was plotted as a function of time (see Experimental Procedures for details). Nearly 100% of cells formed Rad51 foci after IR in all three cell lines by 3 hr after damage. However, decreased binding to Rad51 in the Brca2P3240L/− cell line correlated with a faster reduction in focus-positive cells compared to the heterozygous control. Conversely, the Brca2T3232A/− cell line showed a slower reduction in foci dissolution.
Mentions: Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells synchronized in G1 and released into S phase were exposed to 3 Gy IR immediately after release and harvested every hour afterwards for analysis. Rad51 focus formation was enumerated in 500 cells for each data point by automated high-content microscopy (Figure 4A). The percentage of cells containing Rad51 foci was plotted as a function of time (Figure 4B). In all cell lines, nearly 100% of cells contained Rad51 foci by 3 hr after IR. However, in the Brca2P3240L/− cell line, there was a faster reduction in the percentage of focus-positive cells relative to wild-type, evident 4–5 hr after IR exposure. The P3240L mutation prevents Rad51 binding to the C-terminal Brca2 motif, suggesting that the loss of the interaction promotes this change. Consistent with this, accelerated dissipation of IR-induced Rad51 foci was also observed in Brca2S3239A/S3239A cells (Figure S6). This notion was further supported by the converse effect of the T3232A mutation, which enhanced Rad51 binding. In Brca2T3232A/− cells, there was a slower reduction in the percentage of focus-positive cells (Figure 4B). Together, these observations suggest that the C-terminal Rad51-binding motif in Brca2 regulates not the assembly of Rad51 foci at sites of DNA damage but rather the rate of their dissolution.

Bottom Line: Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry.Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation.We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK.

ABSTRACT

Background: The Rad51 recombinase assembles on DNA to execute homologous DNA recombination (HR). This process is essential to repair replication-associated genomic lesions before cells enter mitosis, but how it is started and stopped during the cell cycle remains poorly understood. Rad51 assembly is regulated by the breast cancer suppressor Brca2, via its evolutionarily conserved BRC repeats, and a distinct carboxy (C)-terminal motif whose biological function is uncertain. Using "hit-and-run" gene targeting to insert single-codon substitutions into the avian Brca2 locus, we report here a previously unrecognized role for the C-terminal motif.

Results: We show that the avian C-terminal motif is functionally cognate with its human counterpart and identify point mutations that either abolish or enhance Rad51 binding. When these mutations are introduced into Brca2, we find that they affect neither the assembly of Rad51 into nuclear foci on damaged DNA nor DNA repair by HR. Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry. Conversely, the slower disassembly of foci in a point mutant that constitutively binds Rad51 correlates with delayed mitosis. Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation.

Conclusions: We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry. This mechanism may ensure that HR terminates before chromosome segregation. Our findings assign a biological function for the C-terminal Brca2 motif in a mechanism that coordinates DNA repair with the cell cycle.

Show MeSH
Related in: MedlinePlus