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At least 775 records · Page 43Linked to original sources
PubMed2005

Nucleation and growth in one dimension. II. Application to DNA replication kinetics.

Inspired by recent experiments on DNA replication, we apply a one-dimensional nucleation-and-growth model to DNA-replication kinetics, focusing on how to extract the time-dependent nucleation rate I (t) and growth speed v from data. We discuss generic experimental problems: namely, spatial inhomogeneity, measurement noise, and finite-size effects. After evaluating how each of these affects the measurements of I (t) and v , we give guidelines for the design of experiments. These ideas are then discussed in the context of the DNA-replication experiments.

Computer Simulation↗
PubMed1974

Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication.

varphiX174 RF (replicative form) II DNA, labeled in vivo with [methyl-(3)H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA(+) cells during RF replication. [(32)P]dCMP was incorporated into the gaps present in the RF II DNA with [alpha-(32)P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated (32)P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the (32)P-labeled fragments. Gel electrophoresis of the products formed by digestion of the (32)P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA(+) cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during varphiX RF replication.

Centrifugation, Density Gradient↗
PubMed2006

Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae.

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Delta mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

Actins↗
PubMed2003

DNA replicative functions of highly-expressed, codon-optimized human papillomavirus proteins E1 and E2.

Human papillomavirus (HPV) DNA replication requires functional HPV early (E) proteins E1 and E2. To determine the biological activity of HPV 16 E1 and E2 mutant proteins under consideration as vaccine candidates, we developed a sensitive real time PCR assay that monitors HPV origin-of-replication-driven DNA synthesis. The assay was used to determine the DNA replicative functions of highly-expressed, codon-optimized HPV 16 E1 and E2 wild type and mutant proteins in transient transfections. Under the assay conditions, the HPV 16 E1 mutant (W439R, G482D) did not support HPV origin-driven DNA synthesis. In contrast, however, an HPV 16 E2 mutant bearing an E39A substitution, reported previously to be severely compromised for DNA replication, was found to be reduced only two-fold in activity and, therefore, considered not sufficiently inactivated for use in vaccines that depend on endogenous protein expression.

Amino Acid Substitution↗
PubMed1980

Effects of reovirus infection on the spatial and temporal organization of DNA replication in L cells.

DNA fiber autoradiography was used to analyze the spatial and temporal organization of activated initiation sites for DNA replication in mouse L929 cells infected with reovirus type 3 (Dearing strain) and in uninfected control cells. Cells were labeled for 10 min with 3H-thymidine at high specific activity followed by 3 h of low specific activity labeling. Reovirus infection causes no change in the rate of replication fork progression, but increases both the mean distance between activated initiation sites by approximately 30% and the nonrandomness in the spatial distribution of the sites along the DNA fibers. Significant synchronization of initiation in adjacent activated sites was detected on DNA fibers from uninfected cells and from reovirus-infected cells. The mean relative initiation time for pairs of initiation events which had occurred prior to high specific activity labeling did not differ significantly between the infected and uninfected cells. The data are consistent with the interpretation that reovirus infection shuts off initiation sites in a coordinated fashion, possibly by preventing activation of entire clusters of potential initiation sites.

Animals↗
PubMed1976

Inhibition of DNA replication by ultraviolet light.

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10J/m2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers.

DNA Repair↗
PubMed1992

DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.

Animals↗
PubMed1977

Differences in the order of termination of DNA replication in human chromosomes in peripheral blood lymphocytes and skin fibroblasts from the same individual.

A comparison of the sequence of terminal replication of DNA in human chromosomes has been made between skin fibroblasts and peripheral blood lymphocytes from the same adult male individual. The chromosomes were identified by quinacrine-banding, and the order of termination of replication was determined by autoradiography. Chromosomes 4, 5, 8, and 13 were found to terminate DNA replication later in lymphocytes than in fibroblasts, wheres chromosomes 15, 19, 20, and 22 were found to terminate their replication earlier in lymphocytes than in fibroblasts.

Adult↗
PubMed2002

Nuclear IE2 structures are related to viral DNA replication sites during baculovirus infection.

The ie2 gene of Autographa californica multicapsid nuclear polyhedrosis virus is 1 of the 10 baculovirus genes that have been identified as factors involved in viral DNA replication. IE2 is detectable in the nucleus as one of the major early-expressed proteins and exhibits a dynamic localization pattern during the infection cycle (D. Murges, I. Quadt, J. Schröer, and D. Knebel-Mörsdorf, Exp. Cell Res. 264:219-232, 2001). Here, we investigated whether IE2 localized to regions of viral DNA replication. After viral DNA was labeled with bromodeoxyuridine (BrdU), confocal imaging indicated that defined IE2 domains colocalized with viral DNA replication centers as soon as viral DNA replication was detectable. In addition, a subpopulation of IE2 structures colocalized with two further virus-encoded replication factors, late expression factor 3 (LEF-3) and the DNA binding protein (DBP). While DBP and LEF-3 structures always colocalized and enlarged simultaneously with viral DNA replication sites, only those IE2 structures that colocalized with replication sites also colocalized with DBP. Replication and transcription of DNA viruses in association with promyelocytic leukemia protein (PML) oncogenic domains have been observed. By confocal imaging we demonstrated that the human PML colocalized with IE2. Triple staining revealed PML/IE2 domains in the vicinity of viral DNA replication centers, while IE2 alone colocalized with early replication sites, demonstrating that PML structures do not form common domains with viral DNA replication centers. Thus, we conclude that IE2 colocalizes alternately with PML and the sites of viral DNA replication. Small ubiquitin-like modifier SUMO-1 has been implicated in the nuclear distribution of PML. Similar to what was found for mammalian cells, small ubiquitin-like modifiers were recruited to PML domains in infected insect cells, which suggests that IE2 and PML colocalize in conserved cellular domains. In summary, our results support a model for IE2 as part of various functional sites in the nucleus that are connected with viral DNA replication.

Animals↗
PubMed2006

The absence of a DNA replication checkpoint in porcine zygotes.

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

Animals↗
PubMed1994

Fis cannot support oriC DNA replication in vitro.

The effects of the histone-like proteins, HU, IHF, and Fis on DNA replication have been examined in vitro using the oriC and pBR322 DNA replication systems. In the oriC system, although low concentrations of HU and IHF stimulated DNA replication, no stimulation was observed upon the addition of Fis. On the contrary, high concentrations of Fis inhibited oriC DNA replication completely. The inhibitory effect of Fis could be overcome when HU, IHF, or RNA polymerase were also present in the reaction mixtures. Unwinding of duplex DNA at the origin correlated with the elevated level of DNA replication. In contrast, only inhibition, rather than stimulation, was observed when these proteins were added to the pBR322 DNA replication system. Thus, the observed dependence on Fis of oriC DNA replication in vivo cannot be attributed to a positive effect on localized DNA unwinding.

Bacterial Proteins↗
PubMed2006

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family.

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.

Bacteriophage T4↗
PubMed1994

Cip1 inhibits DNA replication but not PCNA-dependent nucleotide excision-repair.

BACKGROUND: DNA that is damaged by ultraviolet (UV) light is repaired predominantly by nucleotide excision-repair, a process requiring the DNA polymerase auxiliary factor PCNA. UV-irradiation also induces the production of Cip1 protein via activation of p53. Cip1 is an inhibitor of the cyclin-dependent kinases, which are required for the cell cycle to proceed through the G1/S-phase transition and initiate DNA replication. Inhibition by Cip1 probably causes the block to initiation of DNA replication that is seen in irradiated cells. Cip1 also directly inhibits the function of PCNA during DNA synthesis. As nucleotide excision-repair requires PCNA, the physiological relevance of PCNA inhibition by Cip1 is currently unclear. RESULTS: We show that nucleotide excision-repair of UV-damaged DNA occurs in extracts of Xenopus eggs, and that this reaction is PCNA-dependent. The repair reaction is not inhibited by Cip1, even when the level of PCNA is reduced 100-fold so that it becomes limiting for DNA repair. By contrast, Cip1 strongly suppresses the function of PCNA in replicative DNA synthesis under these conditions. CONCLUSIONS: Cip1 can potentially inhibit DNA replication in Xenopus egg extracts by inhibiting the cyclin-dependent kinase function required for the initiation of replication forks, and also by inhibiting PCNA function. The inhibition of PCNA is selective for its function in DNA replication, however, as Cip1 does not affect PCNA function in nucleotide excision-repair. The induction of Cip1 in response to DNA damage, therefore, allows repair to continue in the genome under conditions in which replication is severely inhibited.

Animals↗
PubMed1989

Adenovirus subviral particles and cores can support limited DNA replication.

Adenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA-complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn2+ ions for Mg2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6.4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for at least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.

Adenoviruses, Human↗
PubMed1995

DNA replication. Once, and only once.

The preparation for DNA replication initiation is tightly linked to cell-cycle progression, ensuring that replication occurs only once per cycle. The time is ripe for a molecular dissection of the links between the two processes.

Cell Cycle↗
PubMedDate not supplied

Regulation of DNA replication by ATR: signaling in response to DNA intermediates.

The nuclear protein kinase ATR controls S-phase progression in response to DNA damage and replication fork stalling, including damage caused by ultraviolet irradiation, hyperoxia, and replication inhibitors like aphidicolin and hydroxyurea. ATR activation and substrate specificity require the presence of adapter and mediator molecules, ultimately resulting in the downstream inhibition of the S-phase kinases that function to initiate DNA replication at origins of replication. The data reviewed strongly support the hypothesis that ATR is activated in response to persistent RPA-bound single-stranded DNA, a common intermediate of unstressed and damaged DNA replication and metabolism.

Animals↗
PubMed1984

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells.

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97% by aphidicolin at 10 micrograms/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30% and 90% depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90% at 100 micrograms/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 micrograms/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase alpha and a non-alpha DNA polymerase (possibly DNA polymerase beta), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase alpha in UDS favored DNA synthesis in the intranucleosomal region.

Animals↗
PubMed1997

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.

Animals↗