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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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The C-Terminal Motif of GgBrca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding(A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of GgBrca2 and Ser3291 of HsBRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved.(B) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of GgBrca2 (GgB23152 aa–3398 aa) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on GgRad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4).(C and D) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of GgB23152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to GgRad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to GgRad51 under the same conditions.(E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type GgBrca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ-32P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction (∗). The bottom panel shows that CDK1 catalyzes the transfer of 32P radiolabel from [γ-32P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.
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fig1: The C-Terminal Motif of GgBrca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding(A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of GgBrca2 and Ser3291 of HsBRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved.(B) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of GgBrca2 (GgB23152 aa–3398 aa) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on GgRad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4).(C and D) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of GgB23152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to GgRad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to GgRad51 under the same conditions.(E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type GgBrca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ-32P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction (∗). The bottom panel shows that CDK1 catalyzes the transfer of 32P radiolabel from [γ-32P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.

Mentions: Three lines of evidence indicate that the C-terminal Rad51-binding motif in Gallus gallus (Gg) Brca2 is functionally cognate with the corresponding region in Homo sapiens (Hs) BRCA2. First, sequence alignment with ClustalW [32] followed by EMBOSS pairwise-alignment algorithms [33] shows considerable evolutionary conservation (Figure 1A). The candidate CDK phosphorylation sites Thr3232, Ser3239, and Thr3271 of GgBrca2 correspond to Ser3284, Ser3291, and Thr3323 of HsBRCA2 (Figure 1A). They are embedded in a 78-residue sequence spanning avian residues 3208–3285 (corresponding to human residues 3260–3337). This motif in GgBrca2 is 42.3% identical and 66.7% similar to its human counterpart (compared to 34.3% identity and 49.9% similarity over the entire avian and human Brca2 molecules). Significantly, the relative position of other key residues is also preserved. Second, the C-terminal motif of GgBrca2, like the cognate region from HsBRCA2 [26], binds to Rad51 in interphase cell extracts, but not in extracts from nocodazole-arrested cells (Figure 1B, compare lane 4 with lanes 1–3). Third, as in the corresponding human region [26], the loss of GgRad51 binding in nocodazole-arrested avian cells is promoted by CDK phosphorylation. Treatment with the CDK inhibitor roscovitine for 30–120 min [34] prevents the nocodazole-induced release of GgRad51 from the C-terminal region of avian Brca2 [26] (Figure 1B).


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)

The C-Terminal Motif of GgBrca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding(A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of GgBrca2 and Ser3291 of HsBRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved.(B) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of GgBrca2 (GgB23152 aa–3398 aa) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on GgRad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4).(C and D) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of GgB23152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to GgRad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to GgRad51 under the same conditions.(E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type GgBrca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ-32P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction (∗). The bottom panel shows that CDK1 catalyzes the transfer of 32P radiolabel from [γ-32P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.
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fig1: The C-Terminal Motif of GgBrca2 Is Functionally Cognate with Its Human Counterpart in CDK-Regulated Rad51 Binding(A) Protein sequence alignment showing the conserved cyclin-dependent kinase (CDK)-phosphorylated residues flanking Ser3239 of GgBrca2 and Ser3291 of HsBRCA2. Gallus gallus residues used in this study are boxed, and residue numbers are indicated below. Asterisks indicate identical residues, double dots indicate conserved substitutions, and single dots indicate residues that are semiconserved.(B) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation of the myc-tagged construct encoding residues 3152–3398 of GgBrca2 (GgB23152 aa–3398 aa) transfected into DT40 cells. Roscovitine treatment for 30–120 min in asynchronous cells had little effect on GgRad51 binding (lanes 2 and 3). However, roscovitine effectively reversed the reduction in binding induced by nocodazole (compare lanes 5 and 6 with lane 4).(C and D) Western blot analysis of GgRad51 and the myc epitope following immunoprecipitation comparing the wild-type (WT) with S3239A/E or P3240L (C) or T3232A (D) variants of GgB23152 aa–3398 aa transfected into DT40 cells. The S3239A/E and P3240L mutations in the conserved Ser-Pro consensus for CDK phosphorylation abrogated binding to GgRad51 in asynchronous and nocodazole-arrested mitotic cell extracts, whereas the T3232A mutation caused enhanced binding to GgRad51 under the same conditions.(E) Thr3232 and Ser3239 can be phosphorylated in vitro by CDK1. A wild-type GgBrca2 peptide fused with GST (WT, sequence at top) or mutant forms in which Thr3232 (T3232A), Ser3239 (S3239A), or both (T3232A/S3239A) were substituted with Ala were subjected to an in vitro kinase assay in the presence of [γ-32P]ATP. Reaction products were cleaved with thrombin to separate the Brca2 peptides from GST. Silver staining (middle panel) measures the loading of the peptides in each reaction (∗). The bottom panel shows that CDK1 catalyzes the transfer of 32P radiolabel from [γ-32P]ATP to the T3232A or S3239A peptides, but not to the double-mutant T3232A/S3239A peptide. Numbers at bottom indicate relative phosphorylation normalized to the amount of peptide present in each sample.
Mentions: Three lines of evidence indicate that the C-terminal Rad51-binding motif in Gallus gallus (Gg) Brca2 is functionally cognate with the corresponding region in Homo sapiens (Hs) BRCA2. First, sequence alignment with ClustalW [32] followed by EMBOSS pairwise-alignment algorithms [33] shows considerable evolutionary conservation (Figure 1A). The candidate CDK phosphorylation sites Thr3232, Ser3239, and Thr3271 of GgBrca2 correspond to Ser3284, Ser3291, and Thr3323 of HsBRCA2 (Figure 1A). They are embedded in a 78-residue sequence spanning avian residues 3208–3285 (corresponding to human residues 3260–3337). This motif in GgBrca2 is 42.3% identical and 66.7% similar to its human counterpart (compared to 34.3% identity and 49.9% similarity over the entire avian and human Brca2 molecules). Significantly, the relative position of other key residues is also preserved. Second, the C-terminal motif of GgBrca2, like the cognate region from HsBRCA2 [26], binds to Rad51 in interphase cell extracts, but not in extracts from nocodazole-arrested cells (Figure 1B, compare lane 4 with lanes 1–3). Third, as in the corresponding human region [26], the loss of GgRad51 binding in nocodazole-arrested avian cells is promoted by CDK phosphorylation. Treatment with the CDK inhibitor roscovitine for 30–120 min [34] prevents the nocodazole-induced release of GgRad51 from the C-terminal region of avian Brca2 [26] (Figure 1B).

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