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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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Coordinate Regulation of Mitotic Entry with Rad51 Binding by the C-Terminal Brca2 Motif(A) Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells were synchronized with mimosine at the G1/S boundary and released into S phase. Samples collected at the indicated time points were stained with anti-MPM2 and analyzed by flow cytometry. The percentage of MPM2-positive cells is plotted on the vertical axis against time after release from G1/S. Each data point represents a mean ± SEM percentage from three samples. MPM2 staining marks cells entering mitosis, which usually increases 4 hr after release from G1/S in control Brca2+/− parental cells (Figure S7B). In the Brca2P3240L/− cell line, this increase is hastened, whereas conversely, in the Brca2T3232A/− cell line, it is delayed.(B) A hypothetical model for the biological role of the C-terminal Brca2 motif in coordinating DNA repair by HR with mitotic entry. During the G2 phase, phosphorylation of the C-terminal Brca2 motif by CDKs regulates the disassembly of DNA-bound Rad51 complexes (1), a process monitored by a mechanism that is independent of the G2 checkpoint for DNA damage (2). Completed disassembly allows entry into the M phase.
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fig6: Coordinate Regulation of Mitotic Entry with Rad51 Binding by the C-Terminal Brca2 Motif(A) Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells were synchronized with mimosine at the G1/S boundary and released into S phase. Samples collected at the indicated time points were stained with anti-MPM2 and analyzed by flow cytometry. The percentage of MPM2-positive cells is plotted on the vertical axis against time after release from G1/S. Each data point represents a mean ± SEM percentage from three samples. MPM2 staining marks cells entering mitosis, which usually increases 4 hr after release from G1/S in control Brca2+/− parental cells (Figure S7B). In the Brca2P3240L/− cell line, this increase is hastened, whereas conversely, in the Brca2T3232A/− cell line, it is delayed.(B) A hypothetical model for the biological role of the C-terminal Brca2 motif in coordinating DNA repair by HR with mitotic entry. During the G2 phase, phosphorylation of the C-terminal Brca2 motif by CDKs regulates the disassembly of DNA-bound Rad51 complexes (1), a process monitored by a mechanism that is independent of the G2 checkpoint for DNA damage (2). Completed disassembly allows entry into the M phase.

Mentions: To test this notion, Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells synchronously passing through S phase were analyzed hourly for DNA content by propidium iodide staining and concurrently with an anti-MPM2 antibody as a marker for entry into mitosis. Strikingly, in the Brca2P3240L/− mutant, the increase in MPM2-positive cells began 2 hr after release into S phase, rather than at 4–5 hr as in Brca2+/− parental cells, suggesting early entry into mitosis (Figure 6A). Conversely, the index of MPM2 staining was delayed in the Brca2T3232A/− mutant cell line compared to the control. In both cases, Rad51 foci were not present in mitotic cells (data not shown). The altered timing of mitotic entry in the Brca2 mutant cells was statistically significant in pairwise comparisons with the control (p < 0.01, two-way ANOVA). Together, these experiments suggest that Rad51 binding to the C-terminal motif of Brca2 coordinates mitotic entry with the dissolution of Rad51 foci.


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)

Coordinate Regulation of Mitotic Entry with Rad51 Binding by the C-Terminal Brca2 Motif(A) Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells were synchronized with mimosine at the G1/S boundary and released into S phase. Samples collected at the indicated time points were stained with anti-MPM2 and analyzed by flow cytometry. The percentage of MPM2-positive cells is plotted on the vertical axis against time after release from G1/S. Each data point represents a mean ± SEM percentage from three samples. MPM2 staining marks cells entering mitosis, which usually increases 4 hr after release from G1/S in control Brca2+/− parental cells (Figure S7B). In the Brca2P3240L/− cell line, this increase is hastened, whereas conversely, in the Brca2T3232A/− cell line, it is delayed.(B) A hypothetical model for the biological role of the C-terminal Brca2 motif in coordinating DNA repair by HR with mitotic entry. During the G2 phase, phosphorylation of the C-terminal Brca2 motif by CDKs regulates the disassembly of DNA-bound Rad51 complexes (1), a process monitored by a mechanism that is independent of the G2 checkpoint for DNA damage (2). Completed disassembly allows entry into the M phase.
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Related In: Results  -  Collection

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

fig6: Coordinate Regulation of Mitotic Entry with Rad51 Binding by the C-Terminal Brca2 Motif(A) Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells were synchronized with mimosine at the G1/S boundary and released into S phase. Samples collected at the indicated time points were stained with anti-MPM2 and analyzed by flow cytometry. The percentage of MPM2-positive cells is plotted on the vertical axis against time after release from G1/S. Each data point represents a mean ± SEM percentage from three samples. MPM2 staining marks cells entering mitosis, which usually increases 4 hr after release from G1/S in control Brca2+/− parental cells (Figure S7B). In the Brca2P3240L/− cell line, this increase is hastened, whereas conversely, in the Brca2T3232A/− cell line, it is delayed.(B) A hypothetical model for the biological role of the C-terminal Brca2 motif in coordinating DNA repair by HR with mitotic entry. During the G2 phase, phosphorylation of the C-terminal Brca2 motif by CDKs regulates the disassembly of DNA-bound Rad51 complexes (1), a process monitored by a mechanism that is independent of the G2 checkpoint for DNA damage (2). Completed disassembly allows entry into the M phase.
Mentions: To test this notion, Brca2+/−, Brca2P3240L/−, and Brca2T3232A/− cells synchronously passing through S phase were analyzed hourly for DNA content by propidium iodide staining and concurrently with an anti-MPM2 antibody as a marker for entry into mitosis. Strikingly, in the Brca2P3240L/− mutant, the increase in MPM2-positive cells began 2 hr after release into S phase, rather than at 4–5 hr as in Brca2+/− parental cells, suggesting early entry into mitosis (Figure 6A). Conversely, the index of MPM2 staining was delayed in the Brca2T3232A/− mutant cell line compared to the control. In both cases, Rad51 foci were not present in mitotic cells (data not shown). The altered timing of mitotic entry in the Brca2 mutant cells was statistically significant in pairwise comparisons with the control (p < 0.01, two-way ANOVA). Together, these experiments suggest that Rad51 binding to the C-terminal motif of Brca2 coordinates mitotic entry with the dissolution of Rad51 foci.

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