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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 promotes the recruitment of BLM and Exo1 to laser-induced DSBs.A. EGFP-CtIP variants and mRFP-PCNA were co-expressed in U2OS cells with endogenous CtIP or both CtIP and H2AX silenced by shRNAs. Live-cell imaging of EGFP-CtIP in S-phase cells, marked by PCNA S-phase-associated replication foci [68], was performed following laser-induced microirradiation. Representative cells show the recruitment of EGFP-CtIP WT or indicated mutants to laser-microirradiated disk regions, which are indicated by red circles in the 0 min cell images. Absolute intensity of EGFP-CtIP fluorescence signals at damage sites was determined; error bars, s.d. B and C. Recruitment of mRFP-BLM (B) and Exo1-mRFP (C) to DSBs was monitored in U2OS cells expressing HA-CtIP or Nbs1-Myc variants, with endogenous CtIP or Nbs1 silenced. Representative cells show the recruitment of mRFP-BLM or Exo1-mRFP to microirradiated regions (a disk region for mRFP-BLM marked by a red circle and dots on a line for Exo1-mRFP marked by a red line in 0 min cell images). Absolute intensities of mRFP-BLM or Exo1-mRFP fluorescence signals were determined; error bars, s.d. Western blots show expression of indicated proteins, with Ku70 as a loading control.
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pgen-1003277-g006: ATM-mediated phosphorylation of CtIP promotes the recruitment of BLM and Exo1 to laser-induced DSBs.A. EGFP-CtIP variants and mRFP-PCNA were co-expressed in U2OS cells with endogenous CtIP or both CtIP and H2AX silenced by shRNAs. Live-cell imaging of EGFP-CtIP in S-phase cells, marked by PCNA S-phase-associated replication foci [68], was performed following laser-induced microirradiation. Representative cells show the recruitment of EGFP-CtIP WT or indicated mutants to laser-microirradiated disk regions, which are indicated by red circles in the 0 min cell images. Absolute intensity of EGFP-CtIP fluorescence signals at damage sites was determined; error bars, s.d. B and C. Recruitment of mRFP-BLM (B) and Exo1-mRFP (C) to DSBs was monitored in U2OS cells expressing HA-CtIP or Nbs1-Myc variants, with endogenous CtIP or Nbs1 silenced. Representative cells show the recruitment of mRFP-BLM or Exo1-mRFP to microirradiated regions (a disk region for mRFP-BLM marked by a red circle and dots on a line for Exo1-mRFP marked by a red line in 0 min cell images). Absolute intensities of mRFP-BLM or Exo1-mRFP fluorescence signals were determined; error bars, s.d. Western blots show expression of indicated proteins, with Ku70 as a loading control.

Mentions: In fission yeast, the interaction of Nbs1 FHA/BRCT domains with the CK2 sites on Ctp1 is required for Ctp1 to be recruited to DSBs [23]. In mammalian cells, CtIP recruitment to DSBs depends on Nbs1 and ATM [29]. To monitor whether CDK- and ATM-mediated phosphorylation regulates CtIP recruitment, we performed live-cell imaging to examine the recruitment of EGFP-tagged CtIP to DSBs upon laser microirradiation. With endogenous CtIP silenced by shRNAs, we showed that the initial recruitments of the CtIP-5A-CDK and CtIP-3A-ATM (S664A/S745A/T859A) mutants to chromosomal DSBs are comparable to CtIP-WT (Figure 6A). However at later time points, CtIP-WT started to disassociate from DSBs faster than the CtIP-5A-CDK and CtIP-3A-ATM mutants, likely due to compromised DSB repair in these mutants. We also monitored CtIP localization to DSB ends in the absence of γH2AX-dependent recruitment to DSB-flanking regions. As expected, inactivation of H2AX significantly reduced CtIP recruitment to laser-generated DSB-containing regions, but γH2AX-independent recruitment of CtIP-5A-CDK and CtIP-3A-ATM mutants to DSB ends was also similar to that of CtIP-WT at the initial stages after laser treatment (Figure 6A). Thus, CDK-phosphorylation of CtIP is not necessarily required for CtIP recruitment to DSBs in mammalian cells.


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 promotes the recruitment of BLM and Exo1 to laser-induced DSBs.A. EGFP-CtIP variants and mRFP-PCNA were co-expressed in U2OS cells with endogenous CtIP or both CtIP and H2AX silenced by shRNAs. Live-cell imaging of EGFP-CtIP in S-phase cells, marked by PCNA S-phase-associated replication foci [68], was performed following laser-induced microirradiation. Representative cells show the recruitment of EGFP-CtIP WT or indicated mutants to laser-microirradiated disk regions, which are indicated by red circles in the 0 min cell images. Absolute intensity of EGFP-CtIP fluorescence signals at damage sites was determined; error bars, s.d. B and C. Recruitment of mRFP-BLM (B) and Exo1-mRFP (C) to DSBs was monitored in U2OS cells expressing HA-CtIP or Nbs1-Myc variants, with endogenous CtIP or Nbs1 silenced. Representative cells show the recruitment of mRFP-BLM or Exo1-mRFP to microirradiated regions (a disk region for mRFP-BLM marked by a red circle and dots on a line for Exo1-mRFP marked by a red line in 0 min cell images). Absolute intensities of mRFP-BLM or Exo1-mRFP fluorescence signals were determined; error bars, s.d. Western blots show expression of indicated proteins, with Ku70 as a loading control.
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pgen-1003277-g006: ATM-mediated phosphorylation of CtIP promotes the recruitment of BLM and Exo1 to laser-induced DSBs.A. EGFP-CtIP variants and mRFP-PCNA were co-expressed in U2OS cells with endogenous CtIP or both CtIP and H2AX silenced by shRNAs. Live-cell imaging of EGFP-CtIP in S-phase cells, marked by PCNA S-phase-associated replication foci [68], was performed following laser-induced microirradiation. Representative cells show the recruitment of EGFP-CtIP WT or indicated mutants to laser-microirradiated disk regions, which are indicated by red circles in the 0 min cell images. Absolute intensity of EGFP-CtIP fluorescence signals at damage sites was determined; error bars, s.d. B and C. Recruitment of mRFP-BLM (B) and Exo1-mRFP (C) to DSBs was monitored in U2OS cells expressing HA-CtIP or Nbs1-Myc variants, with endogenous CtIP or Nbs1 silenced. Representative cells show the recruitment of mRFP-BLM or Exo1-mRFP to microirradiated regions (a disk region for mRFP-BLM marked by a red circle and dots on a line for Exo1-mRFP marked by a red line in 0 min cell images). Absolute intensities of mRFP-BLM or Exo1-mRFP fluorescence signals were determined; error bars, s.d. Western blots show expression of indicated proteins, with Ku70 as a loading control.
Mentions: In fission yeast, the interaction of Nbs1 FHA/BRCT domains with the CK2 sites on Ctp1 is required for Ctp1 to be recruited to DSBs [23]. In mammalian cells, CtIP recruitment to DSBs depends on Nbs1 and ATM [29]. To monitor whether CDK- and ATM-mediated phosphorylation regulates CtIP recruitment, we performed live-cell imaging to examine the recruitment of EGFP-tagged CtIP to DSBs upon laser microirradiation. With endogenous CtIP silenced by shRNAs, we showed that the initial recruitments of the CtIP-5A-CDK and CtIP-3A-ATM (S664A/S745A/T859A) mutants to chromosomal DSBs are comparable to CtIP-WT (Figure 6A). However at later time points, CtIP-WT started to disassociate from DSBs faster than the CtIP-5A-CDK and CtIP-3A-ATM mutants, likely due to compromised DSB repair in these mutants. We also monitored CtIP localization to DSB ends in the absence of γH2AX-dependent recruitment to DSB-flanking regions. As expected, inactivation of H2AX significantly reduced CtIP recruitment to laser-generated DSB-containing regions, but γH2AX-independent recruitment of CtIP-5A-CDK and CtIP-3A-ATM mutants to DSB ends was also similar to that of CtIP-WT at the initial stages after laser treatment (Figure 6A). Thus, CDK-phosphorylation of CtIP is not necessarily required for CtIP recruitment to DSBs in mammalian cells.

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