Replication gaps underlie BRCA-deficiency and therapy response

AbstractCancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism of action of genotoxic chemotherapies.

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Monitoring of DNA Replication and DNA Double-Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis (PFGE)

Methods in Molecular Biology - DNA Electrophoresis ◽

10.1007/978-1-0716-0323-9_11 ◽

2020 ◽

pp. 123-133

Author(s):

Kenji Keyamura ◽

Takashi Hishida

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Gel Electrophoresis ◽

Pulsed Field Gel Electrophoresis ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Pulsed Field

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Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

The Journal of Cell Biology ◽

10.1083/jcb.200905006 ◽

2009 ◽

Vol 187 (1) ◽

pp. 15-23 ◽

Cited By ~ 52

Author(s):

Kevin D. Smith ◽

Michael A. Fu ◽

Eric J. Brown

Keyword(s):

Dna Synthesis ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nucleotide Incorporation ◽

Pathway Activation ◽

Maintain Genome Stability

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

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

Cell Cycle ◽

10.4161/cc.23719 ◽

2013 ◽

Vol 12 (5) ◽

pp. 773-782 ◽

Cited By ~ 35

Author(s):

Tsutomu Shimura ◽

Yasushi Ochiai ◽

Naoto Noma ◽

Toshiyuki Oikawa ◽

Yui Sano ◽

...

Keyword(s):

Dna Replication ◽

Cyclin D1 ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

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Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks

Oncogene ◽

10.1038/sj.onc.1207491 ◽

2004 ◽

Vol 23 (21) ◽

pp. 3872-3882 ◽

Cited By ~ 155

Author(s):

Yun-Gui Yang ◽

Ulrich Cortes ◽

Srinivas Patnaik ◽

Maria Jasin ◽

Zhao-Qi Wang

Keyword(s):

Double Strand Breaks ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Stalled Replication Forks ◽

Parp 1

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DNA Double-Strand Breaks Induced in Human Cells by Twelve Metallic Species: Quantitative Inter-Comparisons and Influence of the ATM Protein

Biomolecules ◽

10.3390/biom11101462 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1462

Author(s):

Muriel Viau ◽

Laurène Sonzogni ◽

Mélanie L. Ferlazzo ◽

Elise Berthel ◽

Sandrine Pereira ◽

...

Keyword(s):

Mechanistic Model ◽

Biological Response ◽

Genotoxic Stress ◽

Double Strand Breaks ◽

Metal Species ◽

Tissue Type ◽

Metal Exposure ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Atm Protein

Despite a considerable amount of data, the molecular and cellular bases of the toxicity due to metal exposure remain unknown. Recent mechanistic models from radiobiology have emerged, pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition and the repair of DNA double-strand breaks (DSB) and the final response to genotoxic stress. In order to document the role of ATM-dependent DSB repair and signalling after metal exposure, we applied twelve different metal species representing nine elements (Al, Cu, Zn Ni, Pd, Cd, Pb, Cr, and Fe) to human skin, mammary, and brain cells. Our findings suggest that metals may directly or indirectly induce DSB at a rate that depends on the metal properties and concentration, and tissue type. At specific metal concentration ranges, the nucleo-shuttling of ATM can be delayed which impairs DSB recognition and repair and contributes to toxicity and carcinogenicity. Interestingly, as observed after low doses of ionizing radiation, some phenomena equivalent to the biological response observed at high metal concentrations may occur at lower concentrations. A general mechanistic model of the biological response to metal exposure based on the nucleo-shuttling of ATM is proposed to describe the metal-induced stress response and to define quantitative endpoints for toxicity and carcinogenicity.

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Illegitimate Recombination Induced by Overproduction of DnaB Helicase in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.181.15.4549-4553.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4549-4553 ◽

Cited By ~ 10

Author(s):

Teruhito Yamashita ◽

Katsuhiro Hanada ◽

Mihoko Iwasaki ◽

Hirotaka Yamaguchi ◽

Hideo Ikeda

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Low Frequency ◽

Double Strand Breaks ◽

Illegitimate Recombination ◽

Replication Forks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Damaging Agents ◽

Dnab Helicase

ABSTRACT Illegitimate recombination that usually takes place at a low frequency is greatly enhanced by treatment with DNA-damaging agents. It is thought that DNA double-strand breaks induced by this DNA damage are important for initiation of illegitimate recombination. Here we show that illegitimate recombination is enhanced by overexpression of the DnaB protein in Escherichia coli. The recombination enhanced by DnaB overexpression occurred between short regions of homology. We propose a model for the initiation of illegitimate recombination in which DnaB overexpression may excessively unwind DNA at replication forks and induce double-strand breaks, resulting in illegitimate recombination. The defect in RecQ has a synergistic effect on the increased illegitimate recombination in cells containing the overproduced DnaB protein, implying that DnaB works in the same pathway as RecQ does but that they work at different steps.

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Natural Products as Anti-Cancerous Therapeutic Molecules Targeted towards Topoisomerases

Current Protein and Peptide Science ◽

10.2174/1389203721666200918152511 ◽

2020 ◽

Vol 21 (11) ◽

pp. 1103-1142

Author(s):

Swati Singh ◽

Veda P. Pandey ◽

Kusum Yadav ◽

Anurag Yadav ◽

U. N. Dwivedi

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Dynamics Simulation ◽

Present Review ◽

Cancer Drug ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Therapeutic Molecules ◽

Topo Ii ◽

Derivatives Of

: 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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Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e11-10-0852 ◽

2012 ◽

Vol 23 (6) ◽

pp. 1058-1067 ◽

Cited By ~ 16

Author(s):

Theresa J. Berens ◽

David P. Toczyski

Keyword(s):

Dna Replication ◽

Double Strand Breaks ◽

Sensor Kinase ◽

Replication Forks ◽

Checkpoint Activation ◽

Replication Checkpoint ◽

Strand Breaks ◽

Crucial Step ◽

Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

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Genome integrity and neurogenesis of postnatal hippocampal neural stem/progenitor cells require a unique regulator Filia

Science Advances ◽

10.1126/sciadv.aba0682 ◽

2020 ◽

Vol 6 (44) ◽

pp. eaba0682 ◽

Cited By ~ 1

Author(s):

Jingzheng Li ◽

Yafang Shang ◽

Lin Wang ◽

Bo Zhao ◽

Chunli Sun ◽

...

Keyword(s):

Progenitor Cells ◽

Neurodevelopmental Disorders ◽

Double Strand Breaks ◽

Genome Integrity ◽

Replication Forks ◽

Proof Of Concept ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Neural Stem Progenitor Cells

Endogenous DNA double-strand breaks (DSBs) formation and repair in neural stem/progenitor cells (NSPCs) play fundamental roles in neurogenesis and neurodevelopmental disorders. NSPCs exhibit heterogeneity in terms of lineage fates and neurogenesis activity. Whether NSPCs also have heterogeneous regulations on DSB formation and repair to accommodate region-specific neurogenesis has not been explored. Here, we identified a regional regulator Filia, which is predominantly expressed in mouse hippocampal NSPCs after birth and regulates DNA DSB formation and repair. On one hand, Filia protects stalling replication forks and prevents the replication stress-associated DNA DSB formation. On the other hand, Filia facilitates the homologous recombination–mediated DNA DSB repair. Consequently, Filia−/− mice had impaired hippocampal NSPC proliferation and neurogenesis and were deficient in learning, memory, and mood regulations. Thus, our study provided the first proof of concept demonstrating the region-specific regulations of DSB formation and repair in subtypes of NSPCs.

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