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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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The interactions of Nbs1 FHA/BRCT domains with CtIP or with MDC1 involve different mechanisms to regulate DSB repair.A. U2OS cells stably expressing control MKO or indicated shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. B. EGFP-HR and EGFP-MMEJ assays were performed with U2OS cells stably expressing MKO or sh-MDC1. Western blotting was performed to show silencing of MDC1, with Ku70 as a loading control. C. EGFP-MMEJ assay was performed in U2OS cells expressing indicated CtIP variants, with endogenous CtIP silenced. Western blotting shows expression of HA-CtIP variants with Ku70 as a loading control. D. EGFP-MMEJ and EGFP-HR assays were performed in U2OS cells expressing Nbs1-WT-Myc or Nbs1-RRHK-Myc mutant with endogenous Nbs1 silenced. Western blotting shows expression of Nbs1-Myc variants with Ku70 as a loading control. E. A model to describe a role for CDK- and ATM-mediated phosphorylation of CtIP in the regulation of end resection and DSB repair (see Discussion).
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pgen-1003277-g007: The interactions of Nbs1 FHA/BRCT domains with CtIP or with MDC1 involve different mechanisms to regulate DSB repair.A. U2OS cells stably expressing control MKO or indicated shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. B. EGFP-HR and EGFP-MMEJ assays were performed with U2OS cells stably expressing MKO or sh-MDC1. Western blotting was performed to show silencing of MDC1, with Ku70 as a loading control. C. EGFP-MMEJ assay was performed in U2OS cells expressing indicated CtIP variants, with endogenous CtIP silenced. Western blotting shows expression of HA-CtIP variants with Ku70 as a loading control. D. EGFP-MMEJ and EGFP-HR assays were performed in U2OS cells expressing Nbs1-WT-Myc or Nbs1-RRHK-Myc mutant with endogenous Nbs1 silenced. Western blotting shows expression of Nbs1-Myc variants with Ku70 as a loading control. E. A model to describe a role for CDK- and ATM-mediated phosphorylation of CtIP in the regulation of end resection and DSB repair (see Discussion).

Mentions: It was described that the Nbs1 FHA/BRCT domains bind to MDC1 through multiple phosphorylated CK2 sites on MDC1 in mammalian cells [42]–[45]. The binding of Nbs1 to multiple CtIP CDK sites shows similar characteristics, where the 5mCDK sites redundantly contribute to the binding. We thus compared the role of Nbs1/MDC1 and Nbs1/CtIP complexes in DSB repair. First, the interaction of the Nbs1 FHA/BRCT domains with phosphorylated CK2 sites on MDC1 is important for Nbs1 to be recruited to DSB-flanking site in a γH2AX-dependent manner [42]–[46]. However, we found that ATM-dependent phosphorylation of CtIP depends on the Nbs1 FHA/BRCT domains, but not on MDC1 and H2AX (Figure 4D and Figure 7A), suggesting that the interactions of the FHA/BRCT domains of Nbs1 with CtIP at CDK sites likely occur directly at DSB ends independently of γH2AX and MDC1 to promote ATM-mediated phosphorylation of CtIP. Therefore, the Nbs1/CtIP and Nbs1/MDC1 complexes are likely formed at different chromosomal locations with respect to the DNA lesions. In addition, overexpression of MDC1 does not influence ATM-mediated phosphorylation of CtIP (Figure S6A), and thus does not affect the interaction of Nbs1 with CtIP, which further supports that the interactions of Nbs1 with CtIP and with MDC1 are independent events. Second, we found that although both Nbs1/MDC1 and Nbs1/CtIP complexes are needed for HR, they play different roles in microhomology-mediated end joining (MMEJ), which is a major pathway of Ku-independent alternative NHEJ (alt-NHEJ, [47], [48]). To monitor MMEJ, we used an EGFP-based repair substrate with 9-bp duplication around the I-Sce1 cleavage site (Figure S6B and [49]). While HR was significantly reduced when MDC1 was inactivated by shRNAs, MMEJ was not affected (Figure 7B). However, when we expressed the CDK mutant CtIP-5A-CDK or CtIP-3A-ATM with endogenous CtIP silenced by shRNAs, both HR and MMEJ were reduced (Figure 7C, Figure 1D and Figure 3A). Consistently, both MMEJ and HR were reduced in the Nbs1 FHA/BRCT domain mutant Nbs1-RRHK with endogenous Nbs1 inactivated by shRNAs (Figure 7D). These data suggest that the associations of the FHA/BRCT domains of Nbs1 with CtIP and with MDC1 are engaged in different mechanisms to regulate DSB repair. While the Nbs1/MDC1 complex is important only for HR, the Nbs1/CtIP complex is needed for both MMEJ and HR.


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)

The interactions of Nbs1 FHA/BRCT domains with CtIP or with MDC1 involve different mechanisms to regulate DSB repair.A. U2OS cells stably expressing control MKO or indicated shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. B. EGFP-HR and EGFP-MMEJ assays were performed with U2OS cells stably expressing MKO or sh-MDC1. Western blotting was performed to show silencing of MDC1, with Ku70 as a loading control. C. EGFP-MMEJ assay was performed in U2OS cells expressing indicated CtIP variants, with endogenous CtIP silenced. Western blotting shows expression of HA-CtIP variants with Ku70 as a loading control. D. EGFP-MMEJ and EGFP-HR assays were performed in U2OS cells expressing Nbs1-WT-Myc or Nbs1-RRHK-Myc mutant with endogenous Nbs1 silenced. Western blotting shows expression of Nbs1-Myc variants with Ku70 as a loading control. E. A model to describe a role for CDK- and ATM-mediated phosphorylation of CtIP in the regulation of end resection and DSB repair (see Discussion).
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Related In: Results  -  Collection

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pgen-1003277-g007: The interactions of Nbs1 FHA/BRCT domains with CtIP or with MDC1 involve different mechanisms to regulate DSB repair.A. U2OS cells stably expressing control MKO or indicated shRNAs were treated with or without IR (10 Gy, recovered for 1 h), lysed, and immunoblotting was performed. B. EGFP-HR and EGFP-MMEJ assays were performed with U2OS cells stably expressing MKO or sh-MDC1. Western blotting was performed to show silencing of MDC1, with Ku70 as a loading control. C. EGFP-MMEJ assay was performed in U2OS cells expressing indicated CtIP variants, with endogenous CtIP silenced. Western blotting shows expression of HA-CtIP variants with Ku70 as a loading control. D. EGFP-MMEJ and EGFP-HR assays were performed in U2OS cells expressing Nbs1-WT-Myc or Nbs1-RRHK-Myc mutant with endogenous Nbs1 silenced. Western blotting shows expression of Nbs1-Myc variants with Ku70 as a loading control. E. A model to describe a role for CDK- and ATM-mediated phosphorylation of CtIP in the regulation of end resection and DSB repair (see Discussion).
Mentions: It was described that the Nbs1 FHA/BRCT domains bind to MDC1 through multiple phosphorylated CK2 sites on MDC1 in mammalian cells [42]–[45]. The binding of Nbs1 to multiple CtIP CDK sites shows similar characteristics, where the 5mCDK sites redundantly contribute to the binding. We thus compared the role of Nbs1/MDC1 and Nbs1/CtIP complexes in DSB repair. First, the interaction of the Nbs1 FHA/BRCT domains with phosphorylated CK2 sites on MDC1 is important for Nbs1 to be recruited to DSB-flanking site in a γH2AX-dependent manner [42]–[46]. However, we found that ATM-dependent phosphorylation of CtIP depends on the Nbs1 FHA/BRCT domains, but not on MDC1 and H2AX (Figure 4D and Figure 7A), suggesting that the interactions of the FHA/BRCT domains of Nbs1 with CtIP at CDK sites likely occur directly at DSB ends independently of γH2AX and MDC1 to promote ATM-mediated phosphorylation of CtIP. Therefore, the Nbs1/CtIP and Nbs1/MDC1 complexes are likely formed at different chromosomal locations with respect to the DNA lesions. In addition, overexpression of MDC1 does not influence ATM-mediated phosphorylation of CtIP (Figure S6A), and thus does not affect the interaction of Nbs1 with CtIP, which further supports that the interactions of Nbs1 with CtIP and with MDC1 are independent events. Second, we found that although both Nbs1/MDC1 and Nbs1/CtIP complexes are needed for HR, they play different roles in microhomology-mediated end joining (MMEJ), which is a major pathway of Ku-independent alternative NHEJ (alt-NHEJ, [47], [48]). To monitor MMEJ, we used an EGFP-based repair substrate with 9-bp duplication around the I-Sce1 cleavage site (Figure S6B and [49]). While HR was significantly reduced when MDC1 was inactivated by shRNAs, MMEJ was not affected (Figure 7B). However, when we expressed the CDK mutant CtIP-5A-CDK or CtIP-3A-ATM with endogenous CtIP silenced by shRNAs, both HR and MMEJ were reduced (Figure 7C, Figure 1D and Figure 3A). Consistently, both MMEJ and HR were reduced in the Nbs1 FHA/BRCT domain mutant Nbs1-RRHK with endogenous Nbs1 inactivated by shRNAs (Figure 7D). These data suggest that the associations of the FHA/BRCT domains of Nbs1 with CtIP and with MDC1 are engaged in different mechanisms to regulate DSB repair. While the Nbs1/MDC1 complex is important only for HR, the Nbs1/CtIP complex is needed for both MMEJ and HR.

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