Sites Of Active Replication Research Articles

Coxsackie B virus.

Coxsackie B virus (CVB) was first isolated in the late 1940s in Coxsackie, New York, and is now known to be widespread worldwide. Infections are common and show some seasonal variation as infections are more common during the summer and early fall. The virus particle is small, nonenveloped, and contains a positive single-stranded RNA genome. The virus attaches to a cellular receptor (often the Coxsackie-adenovirus receptor, CAR) on the cell surface and is then internalized via endocytic vesicles after which the virus uncoats and the RNA genome is delivered across the endosomal membrane into the cytoplasm. Genome translation produces a single polyprotein which is processed into capsid and nonstructural proteins. The nonstructural proteins aid in the production of a negative-strand RNA, which serves as a template for synthesis of new positive-strand RNA molecules, and facilitate virion assembly. Newly synthesized capsid proteins appear in the cytoplasm concomitantly with the appearance of membranous tubulovesicular structures linked to active replication sites. Progeny viruses are typically spread to other cells after lysis of the infected cells, although nonlytic spread has also been reported. The majority of CVB infections are asymptomatic or associated with mild symptoms of the common cold. Occasionally, the CVBs cause severe disease, and infections of neonates can have a fatal outcome.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

γH2AX in the S Phase after UV Irradiation Corresponds to the Sites of DNA Replication and Not DNA Damage

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX — the phosphorylated form of a histone variant H2AX — in the S phase cells within a population. γH2AX has been considered as a definitive marker of DNA damage inside a cell. In this report we show that the γH2AX peak in the S phase after UV irradiation does not report on the extent of DNA damage. Instead it corresponds to the accumulation of γH2AX at the sites of active replication at the time of UV irradiation. We further show that these colocalization sites do not correspond to stalled replication forks — DNA synthesis is still carried on there, albeit at slower speeds. In fact, most of the S phase cells at the time of UV irradiation complete the replication of their DNA and later arrest in the G2 phase of the cell cycle. The study poses new questions as to the roles of γH2AX in the maintenance of genome stability at the replication forks by challenging the conventional idea of γH2AX as simply a mediator and hence a marker of DNA damage and subsequent repair.

Viral load, tissue distribution and histopathological lesions in goats naturally and experimentally infected with the Small Ruminant Lentivirus Genotype E (subtype E1 Roccaverano strain)

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis.Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status.

Analysis of a temperature-sensitive mutation in Uba1: Effects of the click reaction on subsequent immunolabeling of proteins involved in DNA replication

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39°C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3+2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10h after incubation at 39°C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay.

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

Specific Regulation of the Chemokine Response to Viral Hemorrhagic Septicemia Virus at the Entry Site

The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

CD134+ T and B lymphocytes from effector sites of the upper gastrointestinal tract constitute a site of active replication of FIV in chronically infected cats

CD134+ T and B lymphocytes from effector sites of the upper gastrointestinal tract constitute a site of active replication of FIV in chronically infected cats

Interactions of human Cdc45 with the Mcm2–7 complex, the GINS complex, and DNA polymerases δ and ɛ during S phase

Cdc45 is an essential cellular protein that functions in both the initiation and elongation of DNA replication. Here, we analyzed the localization of human Cdc45 and its interactions with other proteins during the cell cycle. Human Cdc45 showed a diffuse distribution in G1 phase, a spot-like pattern in S and G2, and again a diffuse distribution in M phase of the cell cycle. The co-localization of Cdc45 with active replication sites during S phase suggested that the human Cdc45 protein was part of the elongation complex. This view was corroborated by findings that Cdc45 interacted with the elongating DNA polymerases delta and epsilon, with Psf2, which is a component of the GINS complex as well as with Mcm5 and 7, subunits of the putative replicative DNA helicase complex. Hence, Cdc45 may play an important role in elongation of DNA replication by bridging the processive DNA polymerases delta and epsilon with the replicative helicase in the elongating machinery.

Nuclear Localization and Mitogen-activated Protein Kinase Phosphorylationof the Multifunctional ProteinCAD

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.

The Effect of Acyclovir on the Acute and Latent Murine Gammaherpesvirus-68 Infection of Mice

Mice inoculated intranasally with murine gammaherpesvirus-68 were used to evaluate the efficacy of acyclovir (ACV) in the treatment of acute and latent infections. Effectiveness was measured by infectious virus assay of the lung (site of active replication) and infectious centre assay of spleen cells (site of latency). Intraperitoneal administration of ACV at 6-h intervals starting soon after inoculation was more effective in reducing infectious virus in the lung than was treatment with 12-hourly injections commencing 3 days post-infection. Further, ACV treatment during acute infection resulted in an approximately 10-fold reduction in the number of infectious centres in the spleen as compared to placebo-treated animals. However, once latency was established, ACV treatment was not effective in reducing the number of infectious centres in the spleen. This is the first report demonstrating that ACV can be used to minimize the replication of murine gammaherpesvirus in mice at the site of primary infection, resulting in a reduction in the number of latently infected spleen lymphocytes.

Induction and rejoining of radiation-induced DNA single-strand breaks: “tail moment” as a function of position in the cell cycle

Previous results using a variety of methods have shown no significant difference in induction or rejoining of radiation-induced DNA single-strand breaks (SSBs) as a function of cell-cycle position. However, concurrent of DNA damage and cell-cycle position measured using the alkaline comet assay indicates cycle-dependent differences in “tail momen” which has been shown to be a measure of SSBs. Unirradiated S phase cells showed a significantly higher tail moment presumably as a result of the presence of active replication sites. The time required to repair half of the damage appeared to be consistently shorter for G1 cells than for cells in other phases of the cell cycle (3.7 min versus approximately 5 min). Although early kinetics of rejoining (within 5 min) was identical for CHO, V79 and repair-deficient TK6 cells, TK6 cells subsequently repaired fewer breaks than did CHO or V79 cells. S phase TK6 cells rejoined SSBs more slowly than cells in the other phases; differences in rejoining rates could not be explained by the presence of a subset of slowly repairing TK6 cells. We concluide that the comet assay detects subtle differences in the tail moment when cells in different phases of the cell cycle are irradiated and allowed to repair damage. The biological importance of these observations remains to be determined.

Very early replication of scrapie in lymphocytic tissue.

THE incubation period of scrapie in susceptible animals is extremely long1 and its apparently non-viral behaviour is also well known2. Biochemical investigations have usually concentrated on brain tissue when histopathological lesions are maximal3, but it seems important experimentally to locate other sites of active replication. Involvement of lymphocytic tissues has been known for some time4–6 and agent distribution in organs of the mouse after peripheral subcutaneous inoculation suggested a pathogenesis in which the agent localized and multiplied in lymphocytic tissue before being disseminated to sites of secondary replication such as neural cells7. A ten-fold multiplication in the spleen was observed within 8 weeks of inoculation. A genetically controlled delay in the time of an early rise in agent concentration in spleen has been reported after intracerebral or intraperitoneal inoculation8,9.

Studies on the nature of replicating DNA of HeLa cells

Replicating DNA from synchronized HeLa cells pulse labeled with radioactive thymidine differed from non-replicating DNA by its partition to the interphase fraction during extraction with phenol or chloroform. Pulse labeling followed by a chase with non-isotopic thymidine demonstrated that DNA of the interphase fraction was converted to a form which was extractable in the aqueous phase. Blocking DNA synthesis with hydroxyurea prevented this conversion. Sonication of the interphase DNA demonstrated that the property causing the extraction of the DNA into the interphase was localized at or near the site of active replication. Centrifugation of the replicating DNA isolated from the phenol-water interphase revealed that the labeled material separated into two fractions: one which sedimented rapidly to the bottom of a sucrose gradient, but floated in CsCl density gradient; and a fraction which sedimented slightly slower than non-replicating DNA in sucrose gradient but exhibited the same buoyant density in CsCl. The rapidly sedimenting material was converted to a slowly sedimenting material with the same buoyant density as non-replicating DNA by heat, alkali, or sonication, but was unaffected by pronase. Evidence for attachment of a cellular component to replicating DNA is discussed.