Replication occurs at discrete foci spaced throughout nuclei replicating in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 471-477 ◽  
Author(s):  
A.D. Mills ◽  
J.J. Blow ◽  
J.G. White ◽  
W.B. Amos ◽  
D. Wilcock ◽  
...  
Keyword(s):  
Dna Replication ◽  
High Speed ◽  
Microscopic Analysis ◽  
Pulse Labelling ◽  
Replication Forks ◽  
Culture Cells ◽  
Replication Number ◽  
Replication Foci ◽  
Sperm Nuclei

Demembranated Xenopus sperm nuclei were induced to replicate synchronously in a low-speed supernatant (LSS) of Xenopus eggs by preincubation in a high-speed supernatant (HSS). DNA replication was observed by incorporation of [alpha-32P]dATP, BrdUTP or biotin-dUTP. Biotin-dUTP incorporation, visualised with fluorescent streptavidin, reveals a striking pattern of replication foci throughout replicating nuclei. We show that this represents a precursor to the bright uniform fluorescence seen later. Confocal microscopic analysis of nuclei fixed early in replication reveals that these foci of DNA replication number about 100–300 for each nucleus and probably represent the replicon clusters already described for tissue culture cells. Foci are evenly distributed throughout the nuclei and are not concentrated at or near the nuclear envelope. Complete replication of each nucleus occurs in an average time of only one hour in this system. Hence we calculate that there must be at least 300–1000 replication forks together in each cluster. Furthermore, pulse labelling at later times in the period of replication reveals a similar pattern of foci indicating that replication forks remain tightly clustered in groups of at least 300 throughout the period of DNA replication.

Patterns of DNA replication in Drosophila polytene nuclei replicating in Xenopus egg and oocyte extracts

1992 ◽  
Vol 101 (3) ◽  
pp. 509-515
Author(s):  
A.M. Sleeman ◽  
G.H. Leno ◽  
A.D. Mills ◽  
M.P. Fairman ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Salivary Glands ◽  
High Speed ◽  
De Novo ◽  
Replication Forks ◽  
Egg Extract ◽  
Replication Foci ◽  
Sperm Nuclei ◽  
Polytene Nucleus ◽  
Polytene Nuclei

We have used Xenopus laevis cell-free extracts to study patterns of DNA replication in polytene nuclei isolated from salivary glands of Drosophila melanogaster 3rd instar larvae. Replication was visualized by supplementation with biotin-dUTP so that nascent DNA became labelled, thus allowing detection with fluorescein or Texas-Red-conjugated streptavidin. Biotin incorporation was dependent on incubation in extracts. Transverse bands were labelled in high-speed supernatants of eggs or oocytes in which replication does not initiate de novo. These patterns corresponded to the patterns of endogenous replication forks in polytene nuclei, monitored by bromodeoxyuridine incorporation in intact salivary glands. By contrast, when nuclei were incubated in low-speed supernatants of eggs, they underwent more extensive chromatin decondensation and initiated replication. The spatial patterns of replication are strikingly different from the endogenous patterns. Instead they closely resemble patterns of clustered replication forks seen in Xenopus sperm nuclei replicating in the extract. This indicates that the egg extract can impose its pattern of replication foci even when the template is presented in the highly organized form of a polytene nucleus.

DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei

2001 ◽  
Vol 114 (18) ◽  
pp. 3345-3357
Author(s):  
Paola Grandi ◽  
Michail Eltsov ◽  
Inga Nielsen ◽  
Ivan Raška
Keyword(s):  
Dna Replication ◽  
High Speed ◽  
Magnetic Beads ◽  
Protein A ◽  
Restriction Enzymes ◽  
Dna Double Strand Breaks ◽  
Vitro Assay ◽  
Xenopus Egg ◽  
Sperm Nuclei

Replication protein A (RP-A) is involved in DNA replication, repair and recombination. It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase. Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts. Ku86 co-localizes with RP-A in the same foci. An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci. Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA. In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts. Here, also RP-A and Ku cluster in foci as seen through immunofluorescence. Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends. Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment.

scholarly journals Superresolution imaging of individual replication forks reveals unexpected prodrug resistance mechanism

2018 ◽  
Vol 115 (7) ◽  
pp. E1366-E1373 ◽  
Author(s):  
Therese Triemer ◽  
Alessandra Messikommer ◽  
Stella M. K. Glasauer ◽  
Jawad Alzeer ◽  
Miriam H. Paulisch ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Hydroxyl Group ◽  
Nuclear Antigen ◽  
Drug Resistant ◽  
Replication Forks ◽  
Cellular Division ◽  
Apparent Contradiction ◽  
Superresolution Imaging ◽  
Replication Foci

Many drugs require extensive metabolism en route to their targets. High-resolution visualization of prodrug metabolism should therefore utilize analogs containing a small modification that does not interfere with its metabolism or mode of action. In addition to serving as mechanistic probes, such analogs provide candidates for theranostics when applied in both therapeutic and diagnostic modalities. Here a traceable mimic of the widely used anticancer prodrug cytarabine (ara-C) was generated by converting a single hydroxyl group to azide, giving “AzC.” This compound exhibited the same biological profile as ara-C in cell cultures and zebrafish larvae. Using azide-alkyne “click” reactions, we uncovered an apparent contradiction: drug-resistant cells incorporated relatively large quantities of AzC into their genomes and entered S-phase arrest, whereas drug-sensitive cells incorporated only small quantities of AzC. Fluorescence microscopy was used to elucidate structural features associated with drug resistance by characterizing the architectures of stalled DNA replication foci containing AzC, EdU, γH2AX, and proliferating cell nuclear antigen (PCNA). Three-color superresolution imaging revealed replication foci containing one, two, or three partially resolved replication forks. Upon removing AzC from the media, resumption of DNA synthesis and completion of the cell cycle occurred before complete removal of AzC from genomes in vitro and in vivo. These results revealed an important mechanism for the low toxicity of ara-C toward normal tissues and drug-resistant cancer cells, where its efficient incorporation into DNA gives rise to highly stable, stalled replication forks that limit further incorporation of the drug, yet allow for the resumption of DNA synthesis and cellular division following treatment.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

2021 ◽  
Author(s):  
Allison W. McClure ◽  
John F.X. Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

SummaryThe Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

1990 ◽  
Vol 97 (1) ◽  
pp. 177-184
Author(s):  
L.S. Cox ◽  
G.H. Leno
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
High Speed ◽  
De Novo ◽  
Soluble Fraction ◽  
Sperm Chromatin ◽  
Nuclear Assembly ◽  
Egg Extract ◽  
Egg Extracts ◽  
Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kelsey S. Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant D. Schauer ◽  
Stefan H. Mueller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Molecular Machines ◽  
Enzymatic Activities ◽  
Replication Forks ◽  
Dna Substrates ◽  
Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals Phosphorylation of PNKP mediated by CDKs promotes end-processing of Okazaki fragments during DNA replication

2021 ◽  
Author(s):  
Kaima Tsukada ◽  
Rikiya Imamura ◽  
Kotaro Saikawa ◽  
Mizuki Saito ◽  
Naoya Kase ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
High Speed ◽  
Replication Forks ◽  
Cyclin Dependent Kinases ◽  
Okazaki Fragments ◽  
Dna Ligation ◽  
Polynucleotide Kinase ◽  
Dna Ends

Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′ phosphatase and 5′ kinase of DNA ends to promote DNA ligation. Here, we show that PNKP is involved in progression of DNA replication through end-processing of Okazaki fragments (OFs). Cyclin-dependent kinases (CDKs) regulate phosphorylation on threonine 118 (T118) of PNKP, and which phosphorylation allows it to be recruited to OFs. Loss of PNKP and T118 phosphorylation significantly increased unligated OFs and high-speed DNA synthesis in replication forks, suggesting that PNKP T118 phosphorylation is required for OFs ligation for its maturation. Furthermore, phosphatase-dead PNKP also exhibited an accumulation of unligated OFs and high-speed DNA synthesis. Overall, our data suggested that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to OFs and PNKP subsequently promotes end-processing for OFs maturation for stable cell proliferation.

scholarly journals Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Allison McClure ◽  
John Diffley
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genotoxic Stress ◽  
Replication Initiation ◽  
Multiple Roles ◽  
Replication Forks ◽  
Dna Replication Stress ◽  
Necessary And Sufficient

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

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