Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

Background/Aims: Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. Methods: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. Results: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. Conclusion: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.

Download Full-text

Ablating Ku70 phosphorylation results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma

10.1101/2021.03.03.433432 ◽

2021 ◽

Author(s):

Janapriya Saha ◽

Jinsung Bae ◽

Shih-Ya Wang ◽

Lori J. Chappell ◽

Purva Gopal ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Damage ◽

Genomic Instability ◽

Dna Damage Repair ◽

Strand Break ◽

Damage Repair ◽

Dna End Resection ◽

Non Homologous End Joining ◽

Transformed Cell Lines ◽

Dna Ends

SUMMARYMultiple pathways mediate the repair of DNA double-strand break (DSB), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that phosphorylation of the non-homologous end joining (NHEJ) factor, Ku70, is required for the dissociation of the Ku heterodimer from DNA ends to allow DSB repair via homologous recombination (HR). A knock-in mouse, in which phosphorylation is ablated in the three conserved sites of Ku70 (Ku703A/3A), was generated in order to test the hypothesis that Ku70 phosphorylation is required for initiation of HR and that blocking this process results in enhanced genomic instability and tumorigenesis. Here, we show that Ku703A/3A mice develop spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting DNA repair is decreased in these mice. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that phosphorylation-mediated dissociation of Ku heterodimer from DNA ends is required for efficient DNA damage repair and disruption of this process results in genomic instability and accelerated development of HCC.

Download Full-text

GLP-catalyzed H4K16me1 promotes 53BP1 recruitment to permit DNA damage repair and cell survival

Nucleic Acids Research ◽

10.1093/nar/gkz897 ◽

2019 ◽

Vol 47 (21) ◽

pp. 10977-10993 ◽

Cited By ~ 4

Author(s):

Xiaopeng Lu ◽

Ming Tang ◽

Qian Zhu ◽

Qiaoyan Yang ◽

Zhiming Li ◽

...

Keyword(s):

Dna Damage ◽

Cell Survival ◽

Dna Damage Repair ◽

Binding Activity ◽

Critical Event ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Damage Repair ◽

Underlying Mechanisms

Abstract The binding of p53-binding protein 1 (53BP1) to damaged chromatin is a critical event in non-homologous DNA end joining (NHEJ)-mediated DNA damage repair. Although several molecular pathways explaining how 53BP1 binds damaged chromatin have been described, the precise underlying mechanisms are still unclear. Here we report that a newly identified H4K16 monomethylation (H4K16me1) mark is involved in 53BP1 binding activity in the DNA damage response (DDR). During the DDR, H4K16me1 rapidly increases as a result of catalyzation by the histone methyltransferase G9a-like protein (GLP). H4K16me1 shows an increased interaction level with 53BP1, which is important for the timely recruitment of 53BP1 to DNA double-strand breaks. Differing from H4K16 acetylation, H4K16me1 enhances the 53BP1–H4K20me2 interaction at damaged chromatin. Consistently, GLP knockdown markedly attenuates 53BP1 foci formation, leading to impaired NHEJ-mediated repair and decreased cell survival. Together, these data support a novel axis of the DNA damage repair pathway based on H4K16me1 catalysis by GLP, which promotes 53BP1 recruitment to permit NHEJ-mediated DNA damage repair.

Download Full-text

SMCHD1 Promotes ATM-dependent DNA Damage Signaling and Repair of Uncapped Telomeres

10.1101/669119 ◽

2019 ◽

Cited By ~ 1

Author(s):

Aleksandra Vančevska ◽

Verena Pfeiffer ◽

Marianna Feretzaki ◽

Wareed Ahmed ◽

Joachim Lingner

Keyword(s):

Dna Damage ◽

Time Course ◽

End Joining ◽

Checkpoint Signaling ◽

Dna Damage Signaling ◽

Damage Repair ◽

Subsequent Activation ◽

Non Homologous End Joining ◽

Signaling Activation ◽

Structural Maintenance Of Chromosomes

AbstractSMCHD1 (structural maintenance of chromosomes flexible hinge domain containing protein 1) has been implicated in X-chromosome inactivation, imprinting and DNA damage repair. Mutations in SMCHD1 can also cause facioscapulohumoral muscular dystrophy. More recently, SMCHD1 has also been detected as component of telomeric chromatin. Here, we identify requirements of SMCHD1 for DNA damage signaling and non-homologous end joining (NHEJ) at unprotected telomeres. Co-depletion of SMCHD1 with TRF2 reduced the rate of 3’ overhang removal in time course experiments and the number of telomere end fusions. In SMCHD1 deficient cells, the formation of ATM pS1981, γH2AX and 53BP1 containing telomere dysfunction induced foci (TIFs) were diminished indicating defects in checkpoint signaling. Strikingly, removal of TPP1 and subsequent activation of ATR signaling rescued telomere fusion events in TRF2-depleted SMCHD1 knockout cells. Together, these data indicate that SMCHD1 depletion reduces telomere fusions in TRF2-depleted cells due to defects in ATM-dependent DNA checkpoint signaling. SMCHD1 mediates DNA damage signaling activation upstream of ATM phosphorylation at uncapped telomeres.

Download Full-text

LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining

The Journal of Cell Biology ◽

10.1083/jcb.201604112 ◽

2016 ◽

Vol 215 (6) ◽

pp. 801-821 ◽

Cited By ~ 25

Author(s):

Katherine S. Lawrence ◽

Erin C. Tapley ◽

Victor E. Cruz ◽

Qianyan Li ◽

Kayla Aung ◽

...

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Dna Damage Repair ◽

Nonhomologous End Joining ◽

Linc Complex ◽

End Joining ◽

Microtubule Network ◽

Damage Repair ◽

Chromosomal Breaks ◽

Cross Links

The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.

Download Full-text

Defective DNA damage repair leads to frequent catastrophic genomic events in murine and human tumors

10.1101/314518 ◽

2018 ◽

Author(s):

Manasi Ratnaparkhe ◽

John Wong ◽

Pei-Chi Wei ◽

Mario Hlevnjak ◽

Thorsten Kolb ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genomic Rearrangements ◽

End Joining ◽

Catastrophic Events ◽

Mouse Tumors ◽

Damage Repair ◽

Target Dna ◽

Recombination Repair ◽

Non Homologous End Joining

AbstractChromothripsis and chromoanasynthesis are catastrophic events leading to clustered genomic rearrangements. Whole-genome sequencing revealed frequent chromothripsis or chromoanasynthesis (n= 16/26) in brain tumors developing in mice deficient for factors involved in homologous-recombination-repair or non-homologous-end-joining. Catastrophic events were tightly linked to Myc/Mycn amplification, with increased DNA damage and inefficient apoptotic response already observable at early postnatal stages. Inhibition of repair processes and comparison of the mouse tumors with human medulloblastomas (n=68) and glioblastomas (n=32) identified chromothripsis as associated with MYC/MYCN gains and with DNA repair deficiencies, pointing towards therapeutic opportunities to target DNA repair defects in tumors with complex genomic rearrangements.

Download Full-text

OGA is associated with deglycosylation of NONO and the KU complex during DNA damage repair

Cell Death and Disease ◽

10.1038/s41419-021-03910-6 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Yaqi Cui ◽

Rong Xie ◽

Xuefang Zhang ◽

Yi Liu ◽

Yixuan Hu ◽

...

Keyword(s):

Dna Damage ◽

Molecular Mechanisms ◽

Affinity Purification ◽

Dna Damage Repair ◽

Dna Lesions ◽

Deletion Mutants ◽

Biological Functions ◽

End Joining ◽

Protein Affinity ◽

Damage Repair

AbstractAccumulated evidence shows that OGT-mediated O-GlcNAcylation plays an important role in response to DNA damage repair. However, it is unclear if the “eraser” O-GlcNAcase (OGA) participates in this cellular process. Here, we examined the molecular mechanisms and biological functions of OGA in DNA damage repair, and found that OGA was recruited to the sites of DNA damage and mediated deglycosylation following DNA damage. The recruitment of OGA to DNA lesions is mediated by O-GlcNAcylation events. Moreover, we have dissected OGA using deletion mutants and found that C-terminal truncated OGA including the pseudo HAT domain was required for the recruitment of OGA to DNA lesions. Using unbiased protein affinity purification, we found that the pseudo HAT domain was associated with DNA repair factors including NONO and the Ku70/80 complex. Following DNA damage, both NONO and the Ku70/80 complex were O-GlcNAcylated by OGT. The pseudo HAT domain was required to recognize NONO and the Ku70/80 complex for their deglycosylation. Suppression of the deglycosylation prolonged the retention of NONO at DNA lesions and delayed NONO degradation on the chromatin, which impaired non-homologus end joining (NHEJ). Collectively, our study reveals that OGA-mediated deglycosylation plays a key role in DNA damage repair.

Download Full-text