DNA damage‐induced CHK1 autophosphorylation at Ser296 is regulated by an intramolecular mechanism

Okita, Naoyuki; Minato, Shota; Ohmi, Eri; Tanuma, Sei Ichi; Higami, Yoshikazu

doi:10.1016/j.febslet.2012.09.048

Cited by 32 publications

(32 citation statements)

References 39 publications

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“…This apparent induction of the DDR in S phase likely activated a known DNA repair pathway, and in particular ATR, sensitive to DNA replication stress. Indeed, there was activation of ATR following Ajuba depletion, as observed by phosphorylation of Chk1 at residues Ser-345 and Ser-296, which are both ATR-dependent (Liu et al, 2000; Okita et al, 2012) (Figure 3C). In accordance with this finding, p53 was also weakly activated by Ajuba depletion (Figure 3D), as observed by Ser20 phosphorylation associated with siRNA #3.…”

Section: Resultsmentioning

confidence: 94%

LIM Protein Ajuba Participates in the Repression of the ATR-Mediated DNA Damage Response

2013

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LIM proteins constitute a superfamily characterized by the presence of a LIM domain, known to be involved in protein–protein interactions. Our previous work has implicated members of the Zyxin family of LIM proteins, namely TRIP6 and LPP, in the repression of the DNA damage response (DDR) at telomeres. Here, we describe a role for Ajuba, a closely related LIM molecule, in repressing the ATR-mediated DDR. We found that depletion of Ajuba led to apparent delays in the cell cycle, accompanied with increased Rb phosphorylation, Chk1 phosphorylation, induction of p53, and cell death. Ajuba could be found in a complex with replication protein A (RPA), and its depletion led to RPA phosphorylation, known to be an early event in ATR activation. We propose that Ajuba protects against unscheduled ATR signaling by preventing inappropriate RPA phosphorylation.

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Section: Resultsmentioning

confidence: 94%

LIM Protein Ajuba Participates in the Repression of the ATR-Mediated DNA Damage Response

2013

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“…Although ATRi remained active at 8 hour, its effects were significantly different from those of Chk1i. Using Chk1 autophosphorylation at S296 as a functional readout (Okita et al, 2012), we carefully titrated ATRi and Chk1i to determine their respective concentrations required for Chk1 inactivation (Fig. S1A, S4B–C).…”

Section: Resultsmentioning

confidence: 99%

Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

et al. 2015

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The ATR-Chk1 pathway is critical for DNA damage responses and cell cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here, we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.

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“…In response to replication fork stalling, the ATR kinase phosphorylates downstream effector proteins such as Chk1 to initiate checkpoint activation and repair. In response to DNA damage, ATR phosphorylates Chk1 at two main serine residues, S345 and S317, stimulating autophosphorylation at S296 (33,34). To determine if hSSB1 may function in this process, we next depleted cells of hSSB1 and examined phosphorylation of Chk1.…”

Section: Resultsmentioning

confidence: 99%

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Bolderson

Petermann

Croft

et al. 2014

Nucleic Acids Research

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Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

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DNA damage‐induced CHK1 autophosphorylation at Ser296 is regulated by an intramolecular mechanism

Cited by 32 publications

References 39 publications

LIM Protein Ajuba Participates in the Repression of the ATR-Mediated DNA Damage Response

LIM Protein Ajuba Participates in the Repression of the ATR-Mediated DNA Damage Response

Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

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