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Genes Oct 2019Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as... (Review)
Review
Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.
Topics: Animals; Cell Cycle; Cell Cycle Checkpoints; Cell Division; Chromosomes; DNA; DNA Replication; DNA Topoisomerases, Type II; Eukaryotic Cells; Humans; Mitosis; Topoisomerase II Inhibitors
PubMed: 31671531
DOI: 10.3390/genes10110859 -
Nucleic Acids Research Jul 2022Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is...
Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered 'heavy-light' (HL) by incorporation of BrdUTP while unreplicated DNA remains 'light-light' (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.
Topics: DNA; DNA Replication; Genome, Human; Humans; Replication Origin; Sequence Analysis, DNA
PubMed: 35801867
DOI: 10.1093/nar/gkac555 -
Biomolecules Feb 2017DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The... (Review)
Review
DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.
Topics: Animals; Antineoplastic Agents; Cells; DNA Replication; Humans; Stress, Physiological
PubMed: 28230817
DOI: 10.3390/biom7010019 -
The International Journal of... 2016DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or...
DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.
Topics: Animals; Cell-Free System; DNA Barcoding, Taxonomic; DNA Replication; Endonucleases; Fluorescence; Xenopus laevis
PubMed: 27251072
DOI: 10.1387/ijdb.160139oh -
BioEssays : News and Reviews in... Aug 2017The instability of microsatellite DNA repeats is responsible for at least 40 neurodegenerative diseases. Recently, Mirkin and co-workers presented a novel mechanism for... (Review)
Review
The instability of microsatellite DNA repeats is responsible for at least 40 neurodegenerative diseases. Recently, Mirkin and co-workers presented a novel mechanism for microsatellite expansions based on break-induced replication (BIR) at sites of microsatellite-induced replication stalling and fork collapse. The BIR model aims to explain single-step, large expansions of CAG/CTG trinucleotide repeats in dividing cells. BIR has been characterized extensively in Saccharomyces cerevisiae as a mechanism to repair broken DNA replication forks (single-ended DSBs) and degraded telomeric DNA. However, the structural footprints of BIR-like DSB repair have been recognized in human genomic instability and tied to the etiology of diverse developmental diseases; thus, the implications of the paper by Kim et al. (Kim JC, Harris ST, Dinter T, Shah KA, et al., Nat Struct Mol Biol 24: 55-60) extend beyond trinucleotide repeat expansion in yeast and microsatellite instability in human neurological disorders. Significantly, insight into BIR-like repair can explain certain pathways of complex genome rearrangements (CGRs) initiated at non-B form microsatellite DNA in human cancers.
Topics: DNA Repair; DNA Replication; Genomic Instability; Microsatellite Repeats; Recombination, Genetic; Saccharomyces cerevisiae Proteins
PubMed: 28621832
DOI: 10.1002/bies.201700025 -
Nucleic Acids Research Feb 2022The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed...
The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.
Topics: DNA Replication; Genomic Instability; Humans; Replicon; S Phase; Transcription, Genetic
PubMed: 35137218
DOI: 10.1093/nar/gkac069 -
Cytogenetic and Genome Research 2021E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under...
E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.
Topics: Camptothecin; DNA Replication; DNA Replication Timing; Humans; Retinal Pigment Epithelium; Topoisomerase I Inhibitors
PubMed: 34818230
DOI: 10.1159/000518263 -
Biochemical Society Transactions Feb 2019The environmental is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S... (Review)
Review
The environmental is a classical model to study the regulation of the bacterial cell cycle. It divides asymmetrically, giving a stalked cell that immediately enters S phase and a swarmer cell that stays in the G1 phase until it differentiates into a stalked cell. Its genome consists in a single circular chromosome whose replication is tightly regulated so that it happens only in stalked cells and only once cell cycle. Imbalances in chromosomal copy numbers are the most often highly deleterious, if not lethal. This review highlights recent discoveries on pathways that control chromosome replication when is exposed to optimal or less optimal growth conditions. Most of these pathways target two proteins that bind directly onto the chromosomal origin: the highly conserved DnaA initiator of DNA replication and the CtrA response regulator that is found in most The concerted inactivation and proteolysis of CtrA during the swarmer-to-stalked cell transition license cells to enter S phase, while a replisome-associated Regulated Inactivation and proteolysis of DnaA (RIDA) process ensures that initiation starts only once cell cycle. When is stressed, it turns on control systems that delay the G1-to-S phase transition or the elongation of DNA replication, most probably increasing its fitness and adaptation capacities.
Topics: Caulobacter crescentus; Chromosomes, Bacterial; DNA Replication; Gene Expression Regulation, Bacterial; Gram-Negative Bacteria
PubMed: 30626709
DOI: 10.1042/BST20180460 -
Biochimica Et Biophysica Acta May 2010Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the... (Review)
Review
Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.
Topics: Animals; DNA; DNA Replication; Eukaryota; Humans; Multienzyme Complexes; Prokaryotic Cells
PubMed: 19665592
DOI: 10.1016/j.bbapap.2009.07.022 -
Genes & Genetic Systems 2016Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA... (Review)
Review
Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step, resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to secondary strand breaks when they collide with the DNA replication and RNA transcription machinery. DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents. In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to, transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA damage mediated by DNA replication and RNA transcription.
Topics: Camptothecin; DNA Breaks, Single-Stranded; DNA Repair; DNA Replication; DNA Topoisomerases, Type I; Humans; Transcription, Genetic
PubMed: 26616758
DOI: 10.1266/ggs.15-00023