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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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Functional effects of mutating Thr-847 of CtIP. A, alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana; C. elegans, Caenorhabditis elegans; P. nodorum, Phaeosphaeria nodorum; C. globosum, Chaetomium globosum; N. crassa, Neurospora crassa; C. neoformans, Cryptococcus neoformans; Y. lipolytica, Yarrowia lipolytica; A. gossypii, Ashbya gossypii. B, expression levels of GFP-CtIP derivatives in stably transfected clones before (left) or after (right) siRNA depletion of endogenous CtIP (siCtIP). C, representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D, a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ-32P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody (bottom), and phosphorylation was visualized by autoradiography (CDK assay; top).
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fig1: Functional effects of mutating Thr-847 of CtIP. A, alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana; C. elegans, Caenorhabditis elegans; P. nodorum, Phaeosphaeria nodorum; C. globosum, Chaetomium globosum; N. crassa, Neurospora crassa; C. neoformans, Cryptococcus neoformans; Y. lipolytica, Yarrowia lipolytica; A. gossypii, Ashbya gossypii. B, expression levels of GFP-CtIP derivatives in stably transfected clones before (left) or after (right) siRNA depletion of endogenous CtIP (siCtIP). C, representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D, a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ-32P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody (bottom), and phosphorylation was visualized by autoradiography (CDK assay; top).

Mentions: CtIP Function Is Impaired by Mutating Thr-847—A similar CDK consensus sequence to that encompassing Sae2 Ser-267 is present in nearly all Sae2/CtIP orthologues, with the analogous residue of human CtIP being Thr-847 (Fig. 1A). To investigate the potential functions of Thr-847 phosphorylation, we generated stable U2OS cell clones that expressed siRNA-resistant wild-type GFP-tagged CtIP, or siRNA-resistant CtIP derivatives in which Thr-847 was changed to an unphosphorylatable Ala (GFP-CtIP-T847A) or to a negatively charged Glu residue (GFP-CtIP-T847E) to mimic constitutive phosphorylation. We selected for clones that expressed the engineered protein at similar levels to endogenous CtIP (Fig. 1B), and a stable cell line expressing GFP alone was generated as a negative control. As shown in Fig. 1B, transfection with the CtIP siRNA oligonucleotide effectively depleted endogenous CtIP but not the siRNA-resistant GFP-CtIP fusion proteins.


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

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

Functional effects of mutating Thr-847 of CtIP. A, alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana; C. elegans, Caenorhabditis elegans; P. nodorum, Phaeosphaeria nodorum; C. globosum, Chaetomium globosum; N. crassa, Neurospora crassa; C. neoformans, Cryptococcus neoformans; Y. lipolytica, Yarrowia lipolytica; A. gossypii, Ashbya gossypii. B, expression levels of GFP-CtIP derivatives in stably transfected clones before (left) or after (right) siRNA depletion of endogenous CtIP (siCtIP). C, representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D, a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ-32P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody (bottom), and phosphorylation was visualized by autoradiography (CDK assay; top).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666608&req=5

fig1: Functional effects of mutating Thr-847 of CtIP. A, alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana; C. elegans, Caenorhabditis elegans; P. nodorum, Phaeosphaeria nodorum; C. globosum, Chaetomium globosum; N. crassa, Neurospora crassa; C. neoformans, Cryptococcus neoformans; Y. lipolytica, Yarrowia lipolytica; A. gossypii, Ashbya gossypii. B, expression levels of GFP-CtIP derivatives in stably transfected clones before (left) or after (right) siRNA depletion of endogenous CtIP (siCtIP). C, representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D, a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ-32P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody (bottom), and phosphorylation was visualized by autoradiography (CDK assay; top).
Mentions: CtIP Function Is Impaired by Mutating Thr-847—A similar CDK consensus sequence to that encompassing Sae2 Ser-267 is present in nearly all Sae2/CtIP orthologues, with the analogous residue of human CtIP being Thr-847 (Fig. 1A). To investigate the potential functions of Thr-847 phosphorylation, we generated stable U2OS cell clones that expressed siRNA-resistant wild-type GFP-tagged CtIP, or siRNA-resistant CtIP derivatives in which Thr-847 was changed to an unphosphorylatable Ala (GFP-CtIP-T847A) or to a negatively charged Glu residue (GFP-CtIP-T847E) to mimic constitutive phosphorylation. We selected for clones that expressed the engineered protein at similar levels to endogenous CtIP (Fig. 1B), and a stable cell line expressing GFP alone was generated as a negative control. As shown in Fig. 1B, transfection with the CtIP siRNA oligonucleotide effectively depleted endogenous CtIP but not the siRNA-resistant GFP-CtIP fusion proteins.

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