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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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Effects of CtIP mutations on recruitment of proteins to laser-induced damage. Representative images of cells expressing GFP-CtIP variants after laser damage are shown. Cells were immunostained for RPA (magenta) and γH2AX plus cyclin A (red). Damaged cells not expressing cyclin A (G1) and cells positive for cyclin A (S/G2) are marked with empty and filled arrows, respectively.
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fig3: Effects of CtIP mutations on recruitment of proteins to laser-induced damage. Representative images of cells expressing GFP-CtIP variants after laser damage are shown. Cells were immunostained for RPA (magenta) and γH2AX plus cyclin A (red). Damaged cells not expressing cyclin A (G1) and cells positive for cyclin A (S/G2) are marked with empty and filled arrows, respectively.

Mentions: CtIP Mutations Affect Recruitment of CtIP and RPA to DNA Damage—CtIP modulates responses to DNA damage in a cell cycle-dependent manner and is only effectively recruited to sites of laser-induced damage in cyclin A-positive cells (S and G2 cells), when RPA tracts are also formed (11). As CtIP depletion impairs ssDNA formation after camptothecin treatment, this suggests that assessment of camptothecin-induced RPA focus formation would be an effective way to test the effects of mutating CtIP Thr-847. However, this approach has several limitations. First, camptothecin primarily yields DSBs only in S-phase; second, CtIP recruitment into discernible foci is difficult to observe after camptothecin treatment (11); and third, to observe RPA recruitment to sites of DNA damage, cellular preextraction is required, which impedes co-staining for soluble cell cycle markers such as cyclins. To overcome these problems, we used laser DNA-damaging microirradiation. Thus, we laser-irradiated the previously described cell clones stably expressing wild-type or mutated CtIP derivatives after they had been siRNA-depleted of endogenous CtIP. Next, we assessed the appearance of DNA damage tracts by immunofluorescence with antibodies against RPA (to detect RPA-coated ssDNA), γH2AX (to detect damaged chromatin), and cyclin A (to distinguish G1 cells from S/G2 cells. Due to limitations in the number of channels available in the microscope and in the number of non-cross-reacting secondary antibodies, γH2AX and cyclin A were analyzed in the same channel). As shown in Fig. 3, A-D, laser-induced γH2AX tracts were clearly evident in all irradiated cells, irrespective of whether or not they displayed pannuclear cyclin A staining. By contrast, and as reported previously (11), wild-type GFP-CtIP was recruited to damage sites in all S/G2 cells that stained positive for cyclin A (Fig. 3E, filled arrows) but not in cyclin A-negative G1 cells (open arrows). Notably, although a similar recruitment kinetics was observed for the CtIP-T847A mutant (supplemental Fig. 2), RPA recruitment was readily observed in S/G2 cells expressing wild-type CtIP but not in S/G2 cells expressing CtIP-T847A (Fig. 3, I and J), implying that Thr-847 phosphorylation regulates the ability of CtIP to promote resection. In line with this, whereas CtIP-T847E was effectively recruited to damage sites in S/G2 (Fig. 3G, filled arrows; supplemental Fig. 2 for kinetics), it was also recruited to some degree in G1 cells (more than 80% of G1 cells showed weak but visible GFP lines; open arrows). This suggests that mimicking constitutive phosphorylation enhances CtIP activity and raises the possibility that this might allow some CtIP function even in G1 cells. Indeed, cells expressing CtIP-T847E generally displayed more pronounced RPA recruitment than cells expressing wild-type CtIP (Fig. 3, K and I, respectively), and furthermore, weak RPA recruitment was evident in more than 90% of G1 cells expressing CtIP-T847E (Fig. 3K, open arrows), although this was less pronounced than in S/G2 cells (filled arrows). These findings thus suggest that CtIP Thr-847 controls resection during the cell cycle but imply that other CDK-mediated phosphorylations are also needed for optimal resection to occur.


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

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

Effects of CtIP mutations on recruitment of proteins to laser-induced damage. Representative images of cells expressing GFP-CtIP variants after laser damage are shown. Cells were immunostained for RPA (magenta) and γH2AX plus cyclin A (red). Damaged cells not expressing cyclin A (G1) and cells positive for cyclin A (S/G2) are marked with empty and filled arrows, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Effects of CtIP mutations on recruitment of proteins to laser-induced damage. Representative images of cells expressing GFP-CtIP variants after laser damage are shown. Cells were immunostained for RPA (magenta) and γH2AX plus cyclin A (red). Damaged cells not expressing cyclin A (G1) and cells positive for cyclin A (S/G2) are marked with empty and filled arrows, respectively.
Mentions: CtIP Mutations Affect Recruitment of CtIP and RPA to DNA Damage—CtIP modulates responses to DNA damage in a cell cycle-dependent manner and is only effectively recruited to sites of laser-induced damage in cyclin A-positive cells (S and G2 cells), when RPA tracts are also formed (11). As CtIP depletion impairs ssDNA formation after camptothecin treatment, this suggests that assessment of camptothecin-induced RPA focus formation would be an effective way to test the effects of mutating CtIP Thr-847. However, this approach has several limitations. First, camptothecin primarily yields DSBs only in S-phase; second, CtIP recruitment into discernible foci is difficult to observe after camptothecin treatment (11); and third, to observe RPA recruitment to sites of DNA damage, cellular preextraction is required, which impedes co-staining for soluble cell cycle markers such as cyclins. To overcome these problems, we used laser DNA-damaging microirradiation. Thus, we laser-irradiated the previously described cell clones stably expressing wild-type or mutated CtIP derivatives after they had been siRNA-depleted of endogenous CtIP. Next, we assessed the appearance of DNA damage tracts by immunofluorescence with antibodies against RPA (to detect RPA-coated ssDNA), γH2AX (to detect damaged chromatin), and cyclin A (to distinguish G1 cells from S/G2 cells. Due to limitations in the number of channels available in the microscope and in the number of non-cross-reacting secondary antibodies, γH2AX and cyclin A were analyzed in the same channel). As shown in Fig. 3, A-D, laser-induced γH2AX tracts were clearly evident in all irradiated cells, irrespective of whether or not they displayed pannuclear cyclin A staining. By contrast, and as reported previously (11), wild-type GFP-CtIP was recruited to damage sites in all S/G2 cells that stained positive for cyclin A (Fig. 3E, filled arrows) but not in cyclin A-negative G1 cells (open arrows). Notably, although a similar recruitment kinetics was observed for the CtIP-T847A mutant (supplemental Fig. 2), RPA recruitment was readily observed in S/G2 cells expressing wild-type CtIP but not in S/G2 cells expressing CtIP-T847A (Fig. 3, I and J), implying that Thr-847 phosphorylation regulates the ability of CtIP to promote resection. In line with this, whereas CtIP-T847E was effectively recruited to damage sites in S/G2 (Fig. 3G, filled arrows; supplemental Fig. 2 for kinetics), it was also recruited to some degree in G1 cells (more than 80% of G1 cells showed weak but visible GFP lines; open arrows). This suggests that mimicking constitutive phosphorylation enhances CtIP activity and raises the possibility that this might allow some CtIP function even in G1 cells. Indeed, cells expressing CtIP-T847E generally displayed more pronounced RPA recruitment than cells expressing wild-type CtIP (Fig. 3, K and I, respectively), and furthermore, weak RPA recruitment was evident in more than 90% of G1 cells expressing CtIP-T847E (Fig. 3K, open arrows), although this was less pronounced than in S/G2 cells (filled arrows). These findings thus suggest that CtIP Thr-847 controls resection during the cell cycle but imply that other CDK-mediated phosphorylations are also needed for optimal resection to occur.

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