scholarly journals An Antitumor Drug-Induced Topoisomerase Cleavage Complex Blocks a Bacteriophage T4 Replication Fork In Vivo

2000 ◽  
Vol 20 (2) ◽  
pp. 594-603 ◽  
Author(s):  
George Hong ◽  
Kenneth N. Kreuzer
Keyword(s):  
Dna Cleavage ◽  
Cleavage Site ◽  
Replication Fork ◽  
Bacteriophage T4 ◽  
Antitumor Drug ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Drug Induced ◽  
Cleavage Complex

ABSTRACT Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.

scholarly journals Mapping DNA Topoisomerase Binding and Cleavage Genome Wide Using Next-Generation Sequencing Techniques

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 92 ◽  
Author(s):  
Shannon J. McKie ◽  
Anthony Maxwell ◽  
Keir C. Neuman
Keyword(s):  
Next Generation Sequencing ◽  
Dna Cleavage ◽  
Next Generation ◽  
Dna Breaks ◽  
Genome Wide ◽  
Extension Activity ◽  
Nucleotide Resolution ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein–DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
William H. Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

Drug-induced DNA damage and tumor chemosensitivity.

1990 ◽  
Vol 8 (12) ◽  
pp. 2062-2084 ◽  
Author(s):  
R J Epstein
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Tumor Cell ◽  
Malignant Disease ◽  
Dna Lesions ◽  
Drug Induced ◽  
Cell Subpopulations ◽  
Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

scholarly journals Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1566
Author(s):  
Ang Cai ◽  
Ke Bian ◽  
Fangyi Chen ◽  
Qi Tang ◽  
Rachel Carley ◽  
...  
Keyword(s):  
Replication Fork ◽  
Polymerase Activity ◽  
Dna Lesions ◽  
Exact Nature ◽  
Steady State Kinetics ◽  
Modified Dna ◽  
Multiple Conformations ◽  
Cellular Dna

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356–6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

scholarly journals ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

2020 ◽  
Author(s):  
Georgia Chatzinikolaou ◽  
Kalliopi Stratigi ◽  
Kyriacos Agathangelou ◽  
Maria Tsekrekou ◽  
Evi Goulielmaki ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genotoxic Stress ◽  
Gene Promoters ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Genome Wide ◽  
Human Disorders ◽  
Or Gene ◽  
Type Ii Dna Topoisomerases

AbstractType II DNA Topoisomerases (TOP II) generate transient double-strand DNA breaks (DSBs) to resolve topological constraints during transcription. Using genome-wide mapping of DSBs and functional genomics approaches, we show that, in the absence of exogenous genotoxic stress, transcription leads to DSB accumulation and to the recruitment of the structure-specific ERCC1-XPF endonuclease on active gene promoters. Instead, we find that the complex is released from regulatory or gene body elements in UV-irradiated cells. Abrogation of ERCC1 or re-ligation blockage of TOP II-mediated DSBs aggravates the accumulation of transcription-associated γH2Ax and 53BP1 foci, which dissolve when TOP II-mediated DNA cleavage is inhibited. An in vivo biotinylation tagging strategy coupled to a high-throughput proteomics approach reveals that ERCC1-XPF interacts with TOP IIβ and the CTCF/cohesin complex, which co-localize with the heterodimer on DSBs. Together; our findings provide a rational explanation for the remarkable clinical heterogeneity seen in human disorders with ERCC1-XPF defects.

scholarly journals Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

2020 ◽  
Author(s):  
Yann Benureau ◽  
Caroline Pouvelle ◽  
Eliana Moreira Tavares ◽  
Pauline Dupaigne ◽  
Emmanuelle Despras ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Replication Fork ◽  
Damage Tolerance ◽  
Dna Lesions ◽  
Human Cells ◽  
Compensatory Mechanism ◽  
Dna Damage Tolerance ◽  
Replication Intermediate

AbstractDNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

scholarly journals Molecular mechanism for generation of antibody memory

2008 ◽  
Vol 364 (1517) ◽  
pp. 569-575 ◽  
Author(s):  
Velizar Shivarov ◽  
Reiko Shinkura ◽  
Tomomitsu Doi ◽  
Nasim A Begum ◽  
Hitoshi Nagaoka ◽  
...  
Keyword(s):  
Rna Editing ◽  
Dna Cleavage ◽  
De Novo ◽  
Alternative Hypothesis ◽  
Basic Site ◽  
Immunoglobulin Gene ◽  
Dna Breaks ◽  
Dna Deamination

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID–RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo . Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.

scholarly journals Expression and Purification of an Active Form of the Mycobacterium leprae DNA Gyrase and Its Inhibition by Quinolones

2007 ◽  
Vol 51 (5) ◽  
pp. 1643-1648 ◽  
Author(s):  
Stéphanie Matrat ◽  
Stéphanie Petrella ◽  
Emmanuelle Cambau ◽  
Wladimir Sougakoff ◽  
Vincent Jarlier ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Active Form ◽  
Mycobacterium Leprae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Drug Induced ◽  
Vitro Assay ◽  
Drug Concentrations

ABSTRACT Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.

scholarly journals Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo

2020 ◽  
Vol 48 (17) ◽  
pp. 9694-9709
Author(s):  
Zhengping Shao ◽  
Brian J Lee ◽  
Élise Rouleau-Turcotte ◽  
Marie-France Langelier ◽  
Xiaohui Lin ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Live Cell Imaging ◽  
Parp Inhibitors ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Downstream Effector ◽  
Mutation Analyses ◽  
Molecular Nature

Abstract DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as ‘trapping’. To understand the molecular nature of ‘trapping’ in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

scholarly journals Analysis of RuvABC and RecG Involvement in the Escherichia coli Response to the Covalent Topoisomerase-DNA Complex

2010 ◽  
Vol 192 (17) ◽  
pp. 4445-4451 ◽  
Author(s):  
Jeanette H. Sutherland ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Sos Induction ◽  
Cleavage Complex ◽  
Dna Complex

ABSTRACT Topoisomerases form a covalent enzyme-DNA intermediate after initial DNA cleavage. Trapping of the cleavage complex formed by type IIA topoisomerases initiates the bactericidal action of fluoroquinolones. It should be possible also to identify novel antibacterial lead compounds that act with a similar mechanism on type IA bacterial topoisomerases. The cellular response and repair pathways for trapped topoisomerase complexes remain to be fully elucidated. The RuvAB and RecG proteins could play a role in the conversion of the initial protein-DNA complex to double-strand breaks and also in the resolution of the Holliday junction during homologous recombination. Escherichia coli strains with ruvA and recG mutations are found to have increased sensitivity to low levels of norfloxacin treatment, but the mutations had more pronounced effects on survival following the accumulation of covalent complexes formed by mutant topoisomerase I defective in DNA religation. Covalent topoisomerase I and DNA gyrase complexes are converted into double-strand breaks for SOS induction by the RecBCD pathway. SOS induction following topoisomerase I complex accumulation is significantly lower in the ruvA and recG mutants than in the wild-type background, suggesting that RuvAB and RecG may play a role in converting the initial single-strand DNA-protein cleavage complex into a double-strand break prior to repair by homologous recombination. The use of a ruvB mutant proficient in homologous recombination but not in replication fork reversal demonstrated that the replication fork reversal function of RuvAB is required for SOS induction by the covalent complex formed by topoisomerase I.

Sign in / Sign up

Export Citation Format

Share Document