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Human CtIP mediates cell cycle control of DNA end resection and double strand break repair.

Huertas P, Jackson SP - J. Biol. Chem. (2009)

Bottom Line: In G(0) and G(1), DNA double strand breaks are repaired by nonhomologous end joining, whereas in S and G(2), they are also repaired by homologous recombination.Moreover, we show that unlike cells expressing wild-type CtIP, cells expressing the Thr-to-Glu mutant resect DSBs even after CDK inhibition.Finally, we establish that Thr-847 mutations to either Ala or Glu affect DSB repair efficiency, cause hypersensitivity toward DSB-generating agents, and affect the frequency and nature of radiation-induced chromosomal rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge CB2 1QN, United Kingdom.

ABSTRACT
In G(0) and G(1), DNA double strand breaks are repaired by nonhomologous end joining, whereas in S and G(2), they are also repaired by homologous recombination. The human CtIP protein controls double strand break (DSB) resection, an event that occurs effectively only in S/G(2) and that promotes homologous recombination but not non-homologous end joining. Here, we mutate a highly conserved cyclin-dependent kinase (CDK) target motif in CtIP and reveal that mutating Thr-847 to Ala impairs resection, whereas mutating it to Glu to mimic constitutive phosphorylation does not. Moreover, we show that unlike cells expressing wild-type CtIP, cells expressing the Thr-to-Glu mutant resect DSBs even after CDK inhibition. Finally, we establish that Thr-847 mutations to either Ala or Glu affect DSB repair efficiency, cause hypersensitivity toward DSB-generating agents, and affect the frequency and nature of radiation-induced chromosomal rearrangements. These results suggest that CDK-mediated control of resection in human cells operates by mechanisms similar to those recently established in yeast.

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CtIP mutations affect DSB processing. A, cells expressing CtIP variants were treated with DMSO (-) or 25 μm roscovitine (Rosc.)(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations (error bars) of three independent experiments are shown. At least 200 cells were counted per experiment. B, representative images of cells treated in A. C, the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars, standard deviations. D, an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.
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fig4: CtIP mutations affect DSB processing. A, cells expressing CtIP variants were treated with DMSO (-) or 25 μm roscovitine (Rosc.)(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations (error bars) of three independent experiments are shown. At least 200 cells were counted per experiment. B, representative images of cells treated in A. C, the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars, standard deviations. D, an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.

Mentions: CtIP-T847E Promotes Resection Even Following CDK Inhibition—Although the above data suggest that mimicking constitutive phosphorylation of CtIP Thr-847 can partially overcome the CDK requirement for DNA DSB processing, it was formally possible that the cyclin A-negative CtIP-T847E cells displaying RPA recruitment in Fig. 3 were in early S-phase, when CDK was already active, but cyclin A levels were too weak for detection. To address this possibility, we siRNA-depleted endogenous CtIP from cells stably expressing siRNA-resistant GFP-CtIP variants and then treated them with DMSO (negative control) or with the CDK inhibitor, roscovitine. (Supplemental Fig. 1B shows that the fluorescence-activated cell sorter distributions of DMSO- and roscovitine-treated samples were similar, presumably reflecting inhibition of cell cycle transitions by roscovitine.) Next, we treated the cells with X-rays. We chose x-ray treatment because it generates DSBs in all cell cycle phases and allowed us to damage a larger number of cells than we could with laser microirradiation. Subsequently, we assessed cells for DSB formation (γH2AX foci) and ssDNA production (RPA foci). In line with our previous results, DMSO-treated cells expressing wild-type GFP-CtIP or GFP-CtIP-T847E effectively formed RPA foci, whereas cells expressing GFP-CtIP T847A or GFP alone did not (Fig. 4A). Similar results were obtained when we detected ssDNA with an anti-bromodeoxyuridine antibody labeling method (supplemental Fig. 3), indicating that CtIP Thr-847 indeed controls ssDNA formation. Taken together with our other data, these findings therefore indicate that CtIP phosphorylation on Thr-847 controls ssDNA formation and RPA recruitment to sites of damaged DNA induced by camptothecin, laser microirradiation, or X-rays. Furthermore, we found that although roscovitine severely curtailed RPA focus formation following exposure to IR in cells expressing wild-type CtIP, it did not prevent RPA focus formation in cells expressing CtIP-T847E (Fig. 4A). Nevertheless, careful comparisons revealed that roscovitine treatment did reduce the intensity and number of RPA foci in cells expressing CtIP-T847E (Fig. 4, B and C), showing that although CtIP-T847E permits resection even after CDK inhibition, this resection is less extensive than in the presence of CDK activity.


Human CtIP mediates cell cycle control of DNA end resection and double strand break repair.

Huertas P, Jackson SP - J. Biol. Chem. (2009)

CtIP mutations affect DSB processing. A, cells expressing CtIP variants were treated with DMSO (-) or 25 μm roscovitine (Rosc.)(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations (error bars) of three independent experiments are shown. At least 200 cells were counted per experiment. B, representative images of cells treated in A. C, the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars, standard deviations. D, an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: CtIP mutations affect DSB processing. A, cells expressing CtIP variants were treated with DMSO (-) or 25 μm roscovitine (Rosc.)(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations (error bars) of three independent experiments are shown. At least 200 cells were counted per experiment. B, representative images of cells treated in A. C, the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars, standard deviations. D, an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.
Mentions: CtIP-T847E Promotes Resection Even Following CDK Inhibition—Although the above data suggest that mimicking constitutive phosphorylation of CtIP Thr-847 can partially overcome the CDK requirement for DNA DSB processing, it was formally possible that the cyclin A-negative CtIP-T847E cells displaying RPA recruitment in Fig. 3 were in early S-phase, when CDK was already active, but cyclin A levels were too weak for detection. To address this possibility, we siRNA-depleted endogenous CtIP from cells stably expressing siRNA-resistant GFP-CtIP variants and then treated them with DMSO (negative control) or with the CDK inhibitor, roscovitine. (Supplemental Fig. 1B shows that the fluorescence-activated cell sorter distributions of DMSO- and roscovitine-treated samples were similar, presumably reflecting inhibition of cell cycle transitions by roscovitine.) Next, we treated the cells with X-rays. We chose x-ray treatment because it generates DSBs in all cell cycle phases and allowed us to damage a larger number of cells than we could with laser microirradiation. Subsequently, we assessed cells for DSB formation (γH2AX foci) and ssDNA production (RPA foci). In line with our previous results, DMSO-treated cells expressing wild-type GFP-CtIP or GFP-CtIP-T847E effectively formed RPA foci, whereas cells expressing GFP-CtIP T847A or GFP alone did not (Fig. 4A). Similar results were obtained when we detected ssDNA with an anti-bromodeoxyuridine antibody labeling method (supplemental Fig. 3), indicating that CtIP Thr-847 indeed controls ssDNA formation. Taken together with our other data, these findings therefore indicate that CtIP phosphorylation on Thr-847 controls ssDNA formation and RPA recruitment to sites of damaged DNA induced by camptothecin, laser microirradiation, or X-rays. Furthermore, we found that although roscovitine severely curtailed RPA focus formation following exposure to IR in cells expressing wild-type CtIP, it did not prevent RPA focus formation in cells expressing CtIP-T847E (Fig. 4A). Nevertheless, careful comparisons revealed that roscovitine treatment did reduce the intensity and number of RPA foci in cells expressing CtIP-T847E (Fig. 4, B and C), showing that although CtIP-T847E permits resection even after CDK inhibition, this resection is less extensive than in the presence of CDK activity.

Bottom Line: In G(0) and G(1), DNA double strand breaks are repaired by nonhomologous end joining, whereas in S and G(2), they are also repaired by homologous recombination.Moreover, we show that unlike cells expressing wild-type CtIP, cells expressing the Thr-to-Glu mutant resect DSBs even after CDK inhibition.Finally, we establish that Thr-847 mutations to either Ala or Glu affect DSB repair efficiency, cause hypersensitivity toward DSB-generating agents, and affect the frequency and nature of radiation-induced chromosomal rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge CB2 1QN, United Kingdom.

ABSTRACT
In G(0) and G(1), DNA double strand breaks are repaired by nonhomologous end joining, whereas in S and G(2), they are also repaired by homologous recombination. The human CtIP protein controls double strand break (DSB) resection, an event that occurs effectively only in S/G(2) and that promotes homologous recombination but not non-homologous end joining. Here, we mutate a highly conserved cyclin-dependent kinase (CDK) target motif in CtIP and reveal that mutating Thr-847 to Ala impairs resection, whereas mutating it to Glu to mimic constitutive phosphorylation does not. Moreover, we show that unlike cells expressing wild-type CtIP, cells expressing the Thr-to-Glu mutant resect DSBs even after CDK inhibition. Finally, we establish that Thr-847 mutations to either Ala or Glu affect DSB repair efficiency, cause hypersensitivity toward DSB-generating agents, and affect the frequency and nature of radiation-induced chromosomal rearrangements. These results suggest that CDK-mediated control of resection in human cells operates by mechanisms similar to those recently established in yeast.

Show MeSH
Related in: MedlinePlus