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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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Immunoglobulin Gene Diversification in the Mutant Cell Line Brca2S3239A/S3239A Confirms the Integrity of Homologous DNA Recombination(A) Luria-Delbrück fluctuation analysis shows no significant difference in sIgM+ reversion to sIgM− status between the WT and S3239A cell lines. Xrcc2-deficient cells show a significant increase in median fluctuation relative to WT and S3239A (median percentage reversion indicated by horizontal line: WT 0.67%, S3239A 0.57%, Xrcc2 17.6%).(B) Pie charts showing that the source of mutation in VL1 is predominantly gene conversion in WT and Brca2S3239A/S3239A cells but predominantly point mutation in Xrcc2-deficient cells (n = 149, 148, and 54, respectively).(C) Table showing the ratio of point mutation to gene conversion suggests that commitment to homologous DNA recombination (HR) (gene conversion) is intact in Brca2S3239A/S3239A cells but has shifted to nontemplated error-prone repair pathways (point mutation) in Xrcc2-deficient cells.(D) Pie charts showing frequencies of unique gene conversion and point mutation events in WT and Brca2S3239A/S3239A cells indicate a preference for HR over the point mutation used extensively in Xrcc2-deficient cells.
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fig2: Immunoglobulin Gene Diversification in the Mutant Cell Line Brca2S3239A/S3239A Confirms the Integrity of Homologous DNA Recombination(A) Luria-Delbrück fluctuation analysis shows no significant difference in sIgM+ reversion to sIgM− status between the WT and S3239A cell lines. Xrcc2-deficient cells show a significant increase in median fluctuation relative to WT and S3239A (median percentage reversion indicated by horizontal line: WT 0.67%, S3239A 0.57%, Xrcc2 17.6%).(B) Pie charts showing that the source of mutation in VL1 is predominantly gene conversion in WT and Brca2S3239A/S3239A cells but predominantly point mutation in Xrcc2-deficient cells (n = 149, 148, and 54, respectively).(C) Table showing the ratio of point mutation to gene conversion suggests that commitment to homologous DNA recombination (HR) (gene conversion) is intact in Brca2S3239A/S3239A cells but has shifted to nontemplated error-prone repair pathways (point mutation) in Xrcc2-deficient cells.(D) Pie charts showing frequencies of unique gene conversion and point mutation events in WT and Brca2S3239A/S3239A cells indicate a preference for HR over the point mutation used extensively in Xrcc2-deficient cells.

Mentions: It has been suggested from biochemical data that RAD51 binding to the C-terminal region of BRCA2 is necessary for HR [26]. To directly test this idea, we asked whether a mutation that prevents GgRad51 binding to the C terminus of GgBrca2 (Brca2S3239A/S3239A) affects the integrity of gene conversion events in vivo (Figure 2). We monitored mutation in the immunoglobulin gene locus, which constitutively undergoes activation-induced deaminase-dependent hypermutation in the absence of exogenous DNA-damaging agents [31]. The immunoglobulin gene diversification assay is used to determine the rate and mechanism of mutations that render DT40 cells unable to express surface immunoglobulin (sIg), thus reporting events in a specific, endogenous gene locus [39]. In wild-type cells, generation of sIgM− clones from an initially sIgM+ population occurs predominantly through templated mutation from pseudo-V genes (gene conversion, which requires HR). Less frequently, nontemplated mutation (point mutation) may also generate sIgM− clones. DT40 mutants deficient in HR exhibit a shift from templated gene conversion to nontemplated point mutations [29].


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)

Immunoglobulin Gene Diversification in the Mutant Cell Line Brca2S3239A/S3239A Confirms the Integrity of Homologous DNA Recombination(A) Luria-Delbrück fluctuation analysis shows no significant difference in sIgM+ reversion to sIgM− status between the WT and S3239A cell lines. Xrcc2-deficient cells show a significant increase in median fluctuation relative to WT and S3239A (median percentage reversion indicated by horizontal line: WT 0.67%, S3239A 0.57%, Xrcc2 17.6%).(B) Pie charts showing that the source of mutation in VL1 is predominantly gene conversion in WT and Brca2S3239A/S3239A cells but predominantly point mutation in Xrcc2-deficient cells (n = 149, 148, and 54, respectively).(C) Table showing the ratio of point mutation to gene conversion suggests that commitment to homologous DNA recombination (HR) (gene conversion) is intact in Brca2S3239A/S3239A cells but has shifted to nontemplated error-prone repair pathways (point mutation) in Xrcc2-deficient cells.(D) Pie charts showing frequencies of unique gene conversion and point mutation events in WT and Brca2S3239A/S3239A cells indicate a preference for HR over the point mutation used extensively in Xrcc2-deficient cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719694&req=5

fig2: Immunoglobulin Gene Diversification in the Mutant Cell Line Brca2S3239A/S3239A Confirms the Integrity of Homologous DNA Recombination(A) Luria-Delbrück fluctuation analysis shows no significant difference in sIgM+ reversion to sIgM− status between the WT and S3239A cell lines. Xrcc2-deficient cells show a significant increase in median fluctuation relative to WT and S3239A (median percentage reversion indicated by horizontal line: WT 0.67%, S3239A 0.57%, Xrcc2 17.6%).(B) Pie charts showing that the source of mutation in VL1 is predominantly gene conversion in WT and Brca2S3239A/S3239A cells but predominantly point mutation in Xrcc2-deficient cells (n = 149, 148, and 54, respectively).(C) Table showing the ratio of point mutation to gene conversion suggests that commitment to homologous DNA recombination (HR) (gene conversion) is intact in Brca2S3239A/S3239A cells but has shifted to nontemplated error-prone repair pathways (point mutation) in Xrcc2-deficient cells.(D) Pie charts showing frequencies of unique gene conversion and point mutation events in WT and Brca2S3239A/S3239A cells indicate a preference for HR over the point mutation used extensively in Xrcc2-deficient cells.
Mentions: It has been suggested from biochemical data that RAD51 binding to the C-terminal region of BRCA2 is necessary for HR [26]. To directly test this idea, we asked whether a mutation that prevents GgRad51 binding to the C terminus of GgBrca2 (Brca2S3239A/S3239A) affects the integrity of gene conversion events in vivo (Figure 2). We monitored mutation in the immunoglobulin gene locus, which constitutively undergoes activation-induced deaminase-dependent hypermutation in the absence of exogenous DNA-damaging agents [31]. The immunoglobulin gene diversification assay is used to determine the rate and mechanism of mutations that render DT40 cells unable to express surface immunoglobulin (sIg), thus reporting events in a specific, endogenous gene locus [39]. In wild-type cells, generation of sIgM− clones from an initially sIgM+ population occurs predominantly through templated mutation from pseudo-V genes (gene conversion, which requires HR). Less frequently, nontemplated mutation (point mutation) may also generate sIgM− clones. DT40 mutants deficient in HR exhibit a shift from templated gene conversion to nontemplated point mutations [29].

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