Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

: Topoisomerases are reported to resolve the topological problems of DNA during several cellular processes, such as DNA replication, transcription, recombination, and chromatin remodeling. Two types of topoisomerases (Topo I and II) accomplish their designated tasks by introducing single- or double-strand breaks within the duplex DNA molecules, and thus maintain the proper structural conditions of DNA to release the topological torsions, which is generated by unwinding of DNA to access coded information, in the course of replication, transcription, and other processes. Both the topoisomerases have been looked at as crucial targets against various types of cancers such as lung, melanoma, breast, and prostate cancers. Conceptually, targeting topoisomerases will disrupt both DNA replication and transcription, thereby leading to inhibition of cell division and consequently stopping the growth of actively dividing cancerous cells. Since the discovery of camptothecin (an alkaloid) as an inhibitor of Topo I in 1958, a number of derivatives of camptothecin were developed as potent inhibitors of Topo I. Two such derivatives of camptothecin, namely, topotecan and irinotecan, have been commonly used as US Food and Drug Administration (FDA) approved drugs against Topo I. Similarly, the first Topo II inhibitor, namely, etoposide, an analogue of podophyllotoxin, was developed in 1966 and got FDA approval as an anti-cancer drug in 1983. Subsequently, several other inhibitors of Topo II, such as doxorubicin, mitoxantrone, and teniposide, were developed. These drugs have been reported to cause accumulation of cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Thus, the present review describes the anticancer potential of plant-derived secondary metabolites belonging to alkaloids, flavonoids and terpenoids directed against topoisomerases. Furthermore, in view of the recent advances made in the field of computer-aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of these potent anti-cancerous therapeutic molecules.

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miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks

Journal of Biological Chemistry ◽

10.1074/jbc.m116.772392 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3531-3540 ◽

Cited By ~ 15

Author(s):

Baocheng Hu ◽

Xiang Wang ◽

Shuofeng Hu ◽

Xiaomin Ying ◽

Ping Wang ◽

...

Keyword(s):

Cyclin D1 ◽

Opposite Effect ◽

Double Strand Breaks ◽

Human Tumors ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Recombination Repair ◽

Non Homologous End Joining ◽

Novel Target

miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3β exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.

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Chromatin loading of Smc5/6 is induced by DNA replication but not by DNA double-strand breaks

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.10.133 ◽

2006 ◽

Vol 351 (4) ◽

pp. 935-939 ◽

Cited By ~ 9

Author(s):

Takashi Tsuyama ◽

Katsutoshi Inou ◽

Masayuki Seki ◽

Takahiko Seki ◽

Yuji Kumata ◽

...

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Altered DNA ligase activity in human disease

Mutagenesis ◽

10.1093/mutage/gez026 ◽

2019 ◽

Vol 35 (1) ◽

pp. 51-60 ◽

Cited By ~ 3

Author(s):

Alan E Tomkinson ◽

Tasmin Naila ◽

Seema Khattri Bhandari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Double Strand Breaks ◽

Dna Ligase ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Dna Ligases ◽

Ligase Iv ◽

Non Homologous End Joining

Abstract The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

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ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication

Science Advances ◽

10.1126/sciadv.abd9208 ◽

2021 ◽

Vol 7 (25) ◽

pp. eabd9208

Author(s):

Dejie Wang ◽

Jian Ma ◽

Maria Victoria Botuyan ◽

Gaofeng Cui ◽

Yuqian Yan ◽

...

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Nonhomologous End Joining ◽

Genome Integrity ◽

Dna Double Strand Breaks ◽

End Joining ◽

Atm Kinase ◽

Strand Breaks ◽

X Ray ◽

Underlying Mechanisms

53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase–catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

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RecO impedes RecG-SSB binding to impair the strand annealing recombination pathway in E.coli

10.1101/708271 ◽

2019 ◽

Author(s):

Xuefeng Pan ◽

Li Yang ◽

Nan Jiang ◽

Xifang Chen ◽

Bo Li ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Dna Replication ◽

Replication Fork ◽

Double Strand Breaks ◽

Recombinational Repair ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homologous Recombinational Repair ◽

Differential Binding

AbstractFaithful duplication of genomic DNA relies not only on the fidelity of DNA replication itself, but also on fully functional DNA repair and homologous recombination machinery. We report a molecular mechanism responsible for deciding homologous recombinational repair pathways during replication dictated by binding of RecO and RecG to SSB in E.coli. Using a RecG-yfp fusion protein, we found that RecG-yfp foci appeared only in the ΔrecG, ΔrecO and ΔrecA, ΔrecO double mutants. Surprisingly, foci were not observed in wild-type ΔrecG, or double mutants where recG and either recF or, separately recR were deleted. In addition, formation of RecG-yfp foci in the ΔrecO::kanR required wildtype ssb, as ssb-113 could not substitute. This suggests that RecG and RecO binding to SSB is competitive. We also found that the UV resistance of recO alone mutant increased to certain extent by supplementing RecG. In an ssb-113 mutant, RecO and RecG worked following a different pattern. Both RecO and RecG were able to participate in repairing UV damages when grown at permissive temperature, while they could also be involved in making DNA double strand breaks when grown at nonpermissive temperature. So, our results suggested that differential binding of RecG and RecO to SSB in a DNA replication fork in Escherichia coli.may be involved in determining whether the SDSA or DSBR pathway of homologous recombinational repair is used.Author summarySingle strand DNA binding proteins (SSB) stabilize DNA holoenzyme and prevent single strand DNA from folding into non-B DNA structures in a DNA replication fork. It has also been revealed that SSB can also act as a platform for some proteins working in DNA repair and recombination to access DNA molecules when DNA replication fork needs to be reestablished. In Escherichia coli, several proteins working primarily in DNA repair and recombination were found to participate in DNA replication fork resumption by physically interacting with SSB, including RecO and RecG etc. However the hierarchy of these proteins interacting with SSB in Escherichia coli has not been well defined. In this study, we demonstrated a differential binding of RecO and RecG to SSB in DNA replication was used to establish a RecO-dependent pathway of replication fork repair by abolishing a RecG-dependent replication fork repair. We also show that, RecG and RecO could randomly participate in DNA replication repair in the absence of a functional SSB, which may be responsible for the generation of DNA double strand breaks in an ssb-113 mutant in Escherichia coli.

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The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

eLife ◽

10.7554/elife.03297 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 22

Author(s):

Shauna L Houlihan ◽

Yuanyi Feng

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Developmental Disorders ◽

Neural Progenitor ◽

Cortical Layer ◽

S Phase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Genomic Disorders

Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders.

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The Oncogene MYC Triggers Replicative Stress and DNA Damage In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3114.3114 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3114-3114

Author(s):

Francesca Cottini ◽

Teru Hideshima ◽

Giovanni Tonon ◽

Kenneth C. Anderson

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Dna Replication ◽

Plasma Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Replicative Stress ◽

Stress Markers ◽

Dna Replication Stress

Abstract Multiple myeloma (MM) is a clonal proliferation of malignant plasma cells, carrying abnormal karyotypes, chromosomal translocations, and innumerous DNA copy-number variations. We and others have previously shown that MM cells have constitutive DNA damage and DNA damage response (DDR), while normal plasma cells (NPCs) are negative for these DDR markers. Moreover, we recently observed that markers of replicative stress, such as p-ATR and p-CHK1 together with RPA foci, are also present in MM cells. The MYC (or c-MYC) oncogene is pervasively altered in MM. Since MYC is associated with DNA replication stress, oxidative stress, and DDR, we explored whether MYC is implicated in these pathways in MM. Indeed, by analyzing various DNA damage gene expression signatures, we found a positive correlation between MYC levels and ongoing DNA damage. We next examined whether MYC modulation could alter replicative stress markers, and induce DNA double-strand breaks. In a gain-of-function model, c-MYC was expressed in U266 MM cell line, which has low c-MYC levels and importantly shows low levels of ongoing DNA damage. In parallel, the H929 and MM.1S MM cell lines were used to knock-down c-MYC expression. Re-expression of a functional MYC-EGFP in U266 cells induced replicative stress markers, such as RAD51, RPA, and phospho-CHK1 foci, as well as increased RAD51, RPA and phospho-CHK1 protein levels. To determine whether this phenotype was linked to concomitant oxidative stress, we incubated MM cells with an antioxidant reagent N-Acetylcysteine (NAC). We observed a modest reduction in replicative markers after NAC treatment, which was more evident by MYC overexpression. Taken together, these results suggest that the replicative stress induced by MYC is, at least in part, associated with oxidative stress. Additionally, MYC-EGFP positive U266 cells also show DNA damage, evidenced by appearance of phospho-H2A.X foci (which detect DNA double strand breaks), that in turn triggers an intense DNA damage response, assessed by phospho-ATM/phospho CHK2 positivity. In contrast, all these DDR markers were downregulated by MYC silencing, prior to cell death, in MM.1S and H929 MM cell lines. Finally, we examined whether targeting the replicative stress response may represent a novel therapeutic strategy in MM cells with high expression of MYC. Specifically, we treated U266 cells transduced with MYC or control LACZ cells, as well as MM.1S and H929 transfected with a specific MYC-shRNA or their scrambled shRNA controls, with a small molecule ATR inhibitor VE-821 which prevents proper DNA repair after DNA damage. Cells overexpressing MYC were significantly more sensitive to VE-821 treatment compared to controls; conversely MYC-silenced cells were more resistant to VE-821. These results suggest the potential utility of VE-821 as a novel therapeutic agent in cells with high expression of MYC. In conclusion, our data show that MYC may exert its oncogenic activity partly through its ability to trigger DNA replication stress, leading to DNA damage and genomic instability in MM cells. Given the pervasive deregulation of MYC present in MM cells, its role in DNA replication and DNA damage may correlate with the extensive genomic rearrangements observed in MM cells. Therefore, treatment strategies targeting this Achilles' heel may improve patient outcome in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Anderson:Acetylon, Oncopep: Scientific Founder, Scientific Founder Other; Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees.

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Double-strand breaks can induce DNA replication and damage amplification in G2 phase-like oocytes of mice

10.1101/2020.06.29.179010 ◽

2020 ◽

Author(s):

Jun-Yu Ma ◽

Xie Feng ◽

Feng-Yun Xie ◽

Sen Li ◽

Lei-Ning Chen ◽

...

Keyword(s):

Dna Replication ◽

Sequence Similarity ◽

Double Strand Breaks ◽

Small Scale ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Hybrid Mouse ◽

Mouse Oocytes ◽

Chromatin Configuration ◽

G2 Phase

AbstractBreak-induced DNA replication (BIR) have been detected not only in the genome of rare disease patients but also in cancer cells, however, the mechanisms of BIR formation haven’t been explained in details. In the late G2 phase-like mouse oocytes, we found DNA double-strand breaks (DSBs) could induce Rad51 dependent small-scale DNA replication. In addition, we also found the DSBs could be amplified in mouse oocytes, and the amplification could be inhibited by Rad51 inhibitor IBR2 and DNA replication inhibitor ddATP. Lastly, we found the DSB repair was relatively inefficiency in hybrid mouse oocytes compared with that of the purebred mouse oocytes. We found DSBs could induce BIR more easier in hybrid mouse oocytes, indicating the DNA repair in oocytes could be affected by the sequence differences between homologous chromatids. In summary, our results indicated that the condensed chromatin configuration in late G2 phase and the sequence similarity between broken DNA and template DNA are causing factors of BIR in mammalian genome, and the DNA damage could be amplified in late G2 phase cells.

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