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.

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.

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.

scholarly journals Histone H1 Reduces the Frequency of Initiation inXenopus Egg Extract by Limiting the Assembly of Prereplication Complexes on Sperm Chromatin

1998 ◽  
Vol 9 (5) ◽  
pp. 1163-1176 ◽  
Author(s):  
Zhi Hong Lu ◽  
Donald B. Sittman ◽  
Piotr Romanowski ◽  
Gregory H. Leno
Keyword(s):  
Replication Origin ◽  
Histone H1 ◽  
Sperm Chromatin ◽  
Replication Forks ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Interesting Possibility ◽  
Replication Foci ◽  
Xenopus Egg ◽  
Sperm Nuclei

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopussperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.

scholarly journals Mitotic replisome disassembly in vertebrates

2018 ◽  
Author(s):  
Sara Priego Moreno ◽  
Rebecca M. Jones ◽  
Divyasree Poovathumkadavil ◽  
Agnieszka Gambus
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Replication Forks ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Egg Extract ◽  
Eukaryotic Dna ◽  
Higher Eukaryotes

ABSTRACTRecent years have brought a breakthrough in our understanding of the process of eukaryotic DNA replication termination. We have shown that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in S-phase of the cell cycle is driven through polyubiquitylation of one of the replicative helicase subunits Mcm7. Our previous work in C.elegans embryos suggested also an existence of a back-up pathway of replisome disassembly in mitosis. Here we show, that in Xenopus laevis egg extract, any replisome retained on chromatin after S-phase is indeed removed from chromatin in mitosis. This mitotic disassembly pathway depends on formation of K6 and K63 ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and activity of p97/VCP protein segregase. The mitotic replisome pathway is therefore conserved through evolution in higher eukaryotes. However, unlike in lower eukaryotes it does not require SUMO modifications. This process can also remove any helicases from chromatin, including “active” stalled ones, indicating a much wider application of this pathway than just a “back-up” for terminated helicases.

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 Regulation of Gross Chromosomal Rearrangements by Ubiquitin and SUMO Ligases in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (4) ◽  
pp. 1424-1433 ◽  
Author(s):  
Akira Motegi ◽  
Karen Kuntz ◽  
Anju Majeed ◽  
Stephanie Smith ◽  
Kyungjae Myung
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
De Novo ◽  
Chromosomal Rearrangements ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Gross Chromosomal Rearrangements ◽  
Sumo Ligase ◽  
Free Mode ◽  
Proliferating Cell

ABSTRACT Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.

scholarly journals Nuclear Dynamics of PCNA in DNA Replication and Repair

2005 ◽  
Vol 25 (21) ◽  
pp. 9350-9359 ◽  
Author(s):  
Jeroen Essers ◽  
Arjan F. Theil ◽  
Céline Baldeyron ◽  
Wiggert A. van Cappellen ◽  
Adriaan B. Houtsmuller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Residence Time ◽  
Fluorescent Protein ◽  
De Novo ◽  
Dynamic Equilibrium ◽  
Excision Repair ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Replication And Repair ◽  
Replication Foci

ABSTRACT The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

scholarly journals The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.

1991 ◽  
Vol 112 (4) ◽  
pp. 557-566 ◽  
Author(s):  
G H Leno ◽  
R A Laskey
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Fluorescent Intensity ◽  
Free System ◽  
Egg Extract ◽  
Individual Unit ◽  
Egg Extracts ◽  
Replication Foci

We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.

scholarly journals PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

2019 ◽  
Author(s):  
Ke Cong ◽  
Arne Nedergaard Kousholt ◽  
Min Peng ◽  
Nicholas J. Panzarino ◽  
Wei Ting Chelsea Lee ◽  
...  
Keyword(s):  
Dna Replication ◽  
High Speed ◽  
De Novo ◽  
Synthetic Lethality ◽  
Alternative Hypothesis ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
New Paradigm ◽  
Single Stranded Dna ◽  
Synthetic Lethal Interactions

AbstractBRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation. We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions.

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.

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