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The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

Wang H, Shi LZ, Wong CC, Han X, Hwang PY, Truong LN, Zhu Q, Shao Z, Chen DJ, Berns MW, Yates JR, Chen L, Wu X - PLoS Genet. (2013)

Bottom Line: We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1.We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs.Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

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ATM-mediated phosphorylation of CtIP is important for end resection in a CDK-dependent manner.A. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants. B. Clonogenic survival assay after treatment with indicated doses of CPT (1 h) was performed with U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. C. RPA foci formation was examined in U2OS cells with indicated CtIP variants, before or after CPT treatment. The percentage of RPA foci-positive cells was determined as in Figure 1F. D. U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. EGFP-HR repair assay, clonogenic survival and RPA foci formation assays were performed in U2OS cells expressing HA-CtIP-WT or indicated mutants with endogenous CtIP silenced. F. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants.
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pgen-1003277-g003: ATM-mediated phosphorylation of CtIP is important for end resection in a CDK-dependent manner.A. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants. B. Clonogenic survival assay after treatment with indicated doses of CPT (1 h) was performed with U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. C. RPA foci formation was examined in U2OS cells with indicated CtIP variants, before or after CPT treatment. The percentage of RPA foci-positive cells was determined as in Figure 1F. D. U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. EGFP-HR repair assay, clonogenic survival and RPA foci formation assays were performed in U2OS cells expressing HA-CtIP-WT or indicated mutants with endogenous CtIP silenced. F. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants.

Mentions: Consistent with that CDK-mediated phosphorylation of the middle cluster of SP/TP sites is required for ATM to phosphorylate CtIP upon DNA damage, the CDK mutants CtIP-5A-CDK and CtIP-7A-CDK were found to be defective in HR to a similar level as the CtIP-T847A/S889A mutant containing the previously mapped CDK site T847 [17], while the mutants CtIP-S10A/S163A and S549A/S568A did not exhibit such defects (Figure 3A). In addition, the CDK mutants CtIP-5A-CDK and CtIP-12A-CDK were sensitive to CPT (Figure 3B). As revealed by damage-induced RPA foci formation, end resection was defective in the CtIP-5A-CDK mutant to a level comparable to the CtIP-12A-CDK mutant with all CDK sites mutated (Figure 3C), and consequently IR-induced ATR activation as judged by Chk1 phosphorylation was also compromised (Figure 3D). These studies suggest that the 5mCDKs are important for end resection and HR-mediated DSB repair.


The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

Wang H, Shi LZ, Wong CC, Han X, Hwang PY, Truong LN, Zhu Q, Shao Z, Chen DJ, Berns MW, Yates JR, Chen L, Wu X - PLoS Genet. (2013)

ATM-mediated phosphorylation of CtIP is important for end resection in a CDK-dependent manner.A. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants. B. Clonogenic survival assay after treatment with indicated doses of CPT (1 h) was performed with U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. C. RPA foci formation was examined in U2OS cells with indicated CtIP variants, before or after CPT treatment. The percentage of RPA foci-positive cells was determined as in Figure 1F. D. U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. EGFP-HR repair assay, clonogenic survival and RPA foci formation assays were performed in U2OS cells expressing HA-CtIP-WT or indicated mutants with endogenous CtIP silenced. F. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants.
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pgen-1003277-g003: ATM-mediated phosphorylation of CtIP is important for end resection in a CDK-dependent manner.A. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants. B. Clonogenic survival assay after treatment with indicated doses of CPT (1 h) was performed with U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. C. RPA foci formation was examined in U2OS cells with indicated CtIP variants, before or after CPT treatment. The percentage of RPA foci-positive cells was determined as in Figure 1F. D. U2OS cells stably expressing indicated CtIP variants with endogenous CtIP silenced were treated with IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. E. EGFP-HR repair assay, clonogenic survival and RPA foci formation assays were performed in U2OS cells expressing HA-CtIP-WT or indicated mutants with endogenous CtIP silenced. F. EGFP-HR assay was performed with U2OS cells stably expressing HA-CtIP WT or indicated mutants, with endogenous CtIP silenced. Data represent the mean ± s.d. of three independent experiments. Western blot shows expression of HA-CtIP variants.
Mentions: Consistent with that CDK-mediated phosphorylation of the middle cluster of SP/TP sites is required for ATM to phosphorylate CtIP upon DNA damage, the CDK mutants CtIP-5A-CDK and CtIP-7A-CDK were found to be defective in HR to a similar level as the CtIP-T847A/S889A mutant containing the previously mapped CDK site T847 [17], while the mutants CtIP-S10A/S163A and S549A/S568A did not exhibit such defects (Figure 3A). In addition, the CDK mutants CtIP-5A-CDK and CtIP-12A-CDK were sensitive to CPT (Figure 3B). As revealed by damage-induced RPA foci formation, end resection was defective in the CtIP-5A-CDK mutant to a level comparable to the CtIP-12A-CDK mutant with all CDK sites mutated (Figure 3C), and consequently IR-induced ATR activation as judged by Chk1 phosphorylation was also compromised (Figure 3D). These studies suggest that the 5mCDKs are important for end resection and HR-mediated DSB repair.

Bottom Line: We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1.We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs.Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Show MeSH
Related in: MedlinePlus