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Phospho-Chk1 (Ser345) (133D3) Rabbit mAb antibody

RRID:AB_331212

Antibody ID

AB_331212

Target Antigen

Chk1, phospho (Ser345) See NCBI gene human, mouse, rat, monkey

Proper Citation

(Cell Signaling Technology Cat# 2348, RRID:AB_331212)

Clonality

monoclonal antibody

Comments

Applications: W, IF-IC, F. Consolidation: AB_2080326, AB_10079109.

Clone ID

133D3

Host Organism

rabbit

Targeting p38α Increases DNA Damage, Chromosome Instability, and the Anti-tumoral Response to Taxanes in Breast Cancer Cells.

  • Cánovas B
  • Cancer Cell
  • 2018 Jun 11

Literature context:


Abstract:

Breast cancer is the second leading cause of cancer-related death among women. Here we report a role for the protein kinase p38α in coordinating the DNA damage response and limiting chromosome instability during breast tumor progression, and identify the DNA repair regulator CtIP as a p38α substrate. Accordingly, decreased p38α signaling results in impaired ATR activation and homologous recombination repair, with concomitant increases in replication stress, DNA damage, and chromosome instability, leading to cancer cell death and tumor regression. Moreover, we show that pharmacological inhibition of p38α potentiates the effects of taxanes by boosting chromosome instability in murine models and patient-derived xenografts, suggesting the potential interest of combining p38α inhibitors with chemotherapeutic drugs that induce chromosome instability.

Funding information:
  • NIGMS NIH HHS - R01 GM068812(United States)

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

  • Zhu Q
  • Mol. Cell
  • 2018 Jun 7

Literature context:


Abstract:

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.

Funding information:
  • NCI NIH HHS - R01 CA080100()
  • NIAID NIH HHS - R01 AI015066(United States)

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

  • Bellelli R
  • Mol. Cell
  • 2018 May 17

Literature context:


Abstract:

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

Funding information:
  • NIAID NIH HHS - U01 AI070499(United States)

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

  • Chen L
  • Mol. Cell
  • 2018 Feb 1

Literature context:


Abstract:

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Funding information:
  • NIGMS NIH HHS - GM65115(United States)

SLFN11 Blocks Stressed Replication Forks Independently of ATR.

  • Murai J
  • Mol. Cell
  • 2018 Feb 1

Literature context:


Abstract:

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

Funding information:
  • NCI NIH HHS - CA132630(United States)

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

  • Fok KL
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

  • Boege Y
  • Cancer Cell
  • 2017 Sep 11

Literature context:


Abstract:

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Funding information:
  • NIDDK NIH HHS - R01 DK107220()

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

  • Hamperl S
  • Cell
  • 2017 Aug 10

Literature context:


Abstract:

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

Funding information:
  • NIGMS NIH HHS - R01 GM119334()

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

  • Inano S
  • Mol. Cell
  • 2017 Jun 1

Literature context:


Abstract:

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres.

  • Rai R
  • Mol. Cell
  • 2017 Mar 2

Literature context:


Abstract:

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.

Funding information:
  • NCI NIH HHS - P30 CA016359()
  • NIA NIH HHS - R01 AG028888()