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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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Rad51 Foci Are Absent from Mitotic Cells(A) Immunofluorescence analysis of asynchronous WT DT40 cells costained for Rad51 (green) and cyclin B (red). A typical prophase cell (yellow arrowhead) shows intense nuclear (but not cytosolic) staining of cyclin B and is negative for Rad51 foci. DNA is stained with DAPI (blue).(B and C) Immunofluorescence staining of asynchronous human U2OS cells for RAD51 (green) and/or cyclin B (red). RAD51 foci are absent from U2OS cells marked by condensed DAPI-stained chromosomes that have entered mitosis (orange arrowheads).(D) Schematic of the experimental timeline.(E) Cell-cycle profiles of asynchronous, irradiated, and irradiated plus caffeine-treated U2OS cells. DNA content measured by propidium iodide staining and flow cytometry is plotted on the horizontal axis against relative cell number. 2n and 4n peaks represent the G1 and G2/M phases, respectively.(F) Representative immunofluorescence micrographs confirming the absence of RAD51 foci (green) in a mitotic cell (arrowhead) in which γH2AX staining (red) persists after exposure to IR and caffeine. DNA was stained with DAPI (blue). No Rad51 foci were observed in any of 50 mitotic nuclei identified by DAPI staining, whereas γH2AX staining persisted in 38 (76%) of them.
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fig5: Rad51 Foci Are Absent from Mitotic Cells(A) Immunofluorescence analysis of asynchronous WT DT40 cells costained for Rad51 (green) and cyclin B (red). A typical prophase cell (yellow arrowhead) shows intense nuclear (but not cytosolic) staining of cyclin B and is negative for Rad51 foci. DNA is stained with DAPI (blue).(B and C) Immunofluorescence staining of asynchronous human U2OS cells for RAD51 (green) and/or cyclin B (red). RAD51 foci are absent from U2OS cells marked by condensed DAPI-stained chromosomes that have entered mitosis (orange arrowheads).(D) Schematic of the experimental timeline.(E) Cell-cycle profiles of asynchronous, irradiated, and irradiated plus caffeine-treated U2OS cells. DNA content measured by propidium iodide staining and flow cytometry is plotted on the horizontal axis against relative cell number. 2n and 4n peaks represent the G1 and G2/M phases, respectively.(F) Representative immunofluorescence micrographs confirming the absence of RAD51 foci (green) in a mitotic cell (arrowhead) in which γH2AX staining (red) persists after exposure to IR and caffeine. DNA was stained with DAPI (blue). No Rad51 foci were observed in any of 50 mitotic nuclei identified by DAPI staining, whereas γH2AX staining persisted in 38 (76%) of them.

Mentions: We present several lines of evidence that this is indeed the case (Figure S7; Figures 5A–5C). First, in a population of DT40 cells wild-type for Rad51 passing synchronously through the cell cycle (Figure S7A), there was an inverse correlation as they entered mitosis between staining for the mitotic marker anti-MPM2 and the presence of Rad51 foci, suggesting that these markers are mutually exclusive (Figure S7B). Second, we found that individual cells that have entered mitosis rarely contain Rad51 foci. To test this, we costained cells for Rad51 and cyclin B, which becomes nuclear at the onset of nuclear envelope breakdown during prophase [48]. Strikingly, 98% of prophase DT40 cells (n = 100), marked by nuclear cyclin B localization, were negative for Rad51 foci (Figure 5A). The absence of Rad51 foci during mitosis was also a feature in a human cell line. Human U2OS cells in the mitotic prometaphase, metaphase, or anaphase, marked by a typical pattern of DAPI staining, were devoid of RAD51 foci (Figure 5B). As in wild-type DT40 cells, entry into prophase marked by nuclear cyclin B localization was consistently accompanied by the absence of RAD51 foci (Figure 5C). Collectively, these findings suggest that entry into mitosis in both avian and human cells is accompanied by the dissolution of nuclear foci containing RAD51.


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)

Rad51 Foci Are Absent from Mitotic Cells(A) Immunofluorescence analysis of asynchronous WT DT40 cells costained for Rad51 (green) and cyclin B (red). A typical prophase cell (yellow arrowhead) shows intense nuclear (but not cytosolic) staining of cyclin B and is negative for Rad51 foci. DNA is stained with DAPI (blue).(B and C) Immunofluorescence staining of asynchronous human U2OS cells for RAD51 (green) and/or cyclin B (red). RAD51 foci are absent from U2OS cells marked by condensed DAPI-stained chromosomes that have entered mitosis (orange arrowheads).(D) Schematic of the experimental timeline.(E) Cell-cycle profiles of asynchronous, irradiated, and irradiated plus caffeine-treated U2OS cells. DNA content measured by propidium iodide staining and flow cytometry is plotted on the horizontal axis against relative cell number. 2n and 4n peaks represent the G1 and G2/M phases, respectively.(F) Representative immunofluorescence micrographs confirming the absence of RAD51 foci (green) in a mitotic cell (arrowhead) in which γH2AX staining (red) persists after exposure to IR and caffeine. DNA was stained with DAPI (blue). No Rad51 foci were observed in any of 50 mitotic nuclei identified by DAPI staining, whereas γH2AX staining persisted in 38 (76%) of them.
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fig5: Rad51 Foci Are Absent from Mitotic Cells(A) Immunofluorescence analysis of asynchronous WT DT40 cells costained for Rad51 (green) and cyclin B (red). A typical prophase cell (yellow arrowhead) shows intense nuclear (but not cytosolic) staining of cyclin B and is negative for Rad51 foci. DNA is stained with DAPI (blue).(B and C) Immunofluorescence staining of asynchronous human U2OS cells for RAD51 (green) and/or cyclin B (red). RAD51 foci are absent from U2OS cells marked by condensed DAPI-stained chromosomes that have entered mitosis (orange arrowheads).(D) Schematic of the experimental timeline.(E) Cell-cycle profiles of asynchronous, irradiated, and irradiated plus caffeine-treated U2OS cells. DNA content measured by propidium iodide staining and flow cytometry is plotted on the horizontal axis against relative cell number. 2n and 4n peaks represent the G1 and G2/M phases, respectively.(F) Representative immunofluorescence micrographs confirming the absence of RAD51 foci (green) in a mitotic cell (arrowhead) in which γH2AX staining (red) persists after exposure to IR and caffeine. DNA was stained with DAPI (blue). No Rad51 foci were observed in any of 50 mitotic nuclei identified by DAPI staining, whereas γH2AX staining persisted in 38 (76%) of them.
Mentions: We present several lines of evidence that this is indeed the case (Figure S7; Figures 5A–5C). First, in a population of DT40 cells wild-type for Rad51 passing synchronously through the cell cycle (Figure S7A), there was an inverse correlation as they entered mitosis between staining for the mitotic marker anti-MPM2 and the presence of Rad51 foci, suggesting that these markers are mutually exclusive (Figure S7B). Second, we found that individual cells that have entered mitosis rarely contain Rad51 foci. To test this, we costained cells for Rad51 and cyclin B, which becomes nuclear at the onset of nuclear envelope breakdown during prophase [48]. Strikingly, 98% of prophase DT40 cells (n = 100), marked by nuclear cyclin B localization, were negative for Rad51 foci (Figure 5A). The absence of Rad51 foci during mitosis was also a feature in a human cell line. Human U2OS cells in the mitotic prometaphase, metaphase, or anaphase, marked by a typical pattern of DAPI staining, were devoid of RAD51 foci (Figure 5B). As in wild-type DT40 cells, entry into prophase marked by nuclear cyclin B localization was consistently accompanied by the absence of RAD51 foci (Figure 5C). Collectively, these findings suggest that entry into mitosis in both avian and human cells is accompanied by the dissolution of nuclear foci containing RAD51.

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