Multiple Rounds Of DNA Synthesis Research Articles

Flying High-Muscle-Specific Underreplication in Drosophila.

Drosophila underreplicate the DNA of thoracic nuclei, stalling during S phase at a point that is proportional to the total genome size in each species. In polytene tissues, such as the Drosophila salivary glands, all of the nuclei initiate multiple rounds of DNA synthesis and underreplicate. Yet, only half of the nuclei isolated from the thorax stall; the other half do not initiate S phase. Our question was, why half? To address this question, we use flow cytometry to compare underreplication phenotypes between thoracic tissues. When individual thoracic tissues are dissected and the proportion of stalled DNA synthesis is scored in each tissue type, we find that underreplication occurs in the indirect flight muscle, with the majority of underreplicated nuclei in the dorsal longitudinal muscles (DLM). Half of the DNA in the DLM nuclei stall at S phase between the unreplicated G0 and fully replicated G1. The dorsal ventral flight muscle provides the other source of underreplication, and yet, there, the replication stall point is earlier (less DNA replicated), and the endocycle is initiated. The differences in underreplication and ploidy in the indirect flight muscles provide a new tool to study heterochromatin, underreplication and endocycle control.

Plant Cyclin-Dependent Kinase Inhibitors of the KRP Family: Potent Inhibitors of Root-Knot Nematode Feeding Sites in Plant Roots.

Root-knot nematodes (RKN), Meloidogyne spp., are distributed worldwide and impose severe economic damage to many agronomically important crops. The plant cell cycle machinery is considered one of the pivotal components for the formation of nematode feeding sites (NFSs) or galls. These feeding sites contain five to nine hypertrophied giant cells (GC) resulting from developmental reprogramming of host root cells by this pathogen. GC undergo synchronous waves of mitotic activity uncoupled from cytokinesis giving rise to large multinucleate cells. As development of the NFS progresses, multiple rounds of DNA synthesis occur in the nuclei of GC, coupled with nuclear and cellular expansion. These cells are highly metabolically active and provide the nematode with nutrients necessary for its development and completion of its life cycle. In Arabidopsis seven cyclin dependent kinase inhibitors (CKIs) belonging to the interactors/inhibitors of the cyclin dependent kinases (ICK) family, also referred as Kip-Related Proteins (KRPs) have been identified. Interactions of KRPs with CDK/Cyclin complexes decrease CDK activity, affecting both cell cycle progression and DNA content in a concentration-dependent manner. We performed the functional analysis of all Arabidopsis KRP gene members during RKN interaction in Arabidopsis to obtain more insight into their role during gall development. We demonstrated that three members of this family (KRP2, KRP5, and KRP6) were highly expressed in galls and were important for cell cycle regulation during NFS development as shown by their different modes of action. We also pointed out that cell cycle inhibition through overexpression of all members of the KRP family can affect NFS development and consequently compromise the nematode’s life cycle. In this review we summarized our recent understanding of the KRP family of genes, and their role in controlling cell cycle progression at the RKN feeding site.

Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis

The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.

Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.

The HIV-1 Tat Protein Stimulates Reverse Transcription In Vitro

The role of Tat in HIV-1 reverse transcription has been controversial largely because different studies have observed disparate effects of the Tat protein on reverse transcription. Studies of HIV-1 lacking a functional tat gene demonstrated a decrease in reverse transcription efficiency following infection of T-cells however, in vitro recombinant Tat(1-86) has been shown to inhibit RT activity. Here we show that 20-200 nM of both N-terminally histidine-tagged recombinant Tat(1-72) and Tat(1-86) stimulated reverse transcription by HIV-1 reverse transcriptase (RT) in vitro by 2-3 fold. However, both Tat species were efficient inhibitors of RT activity at 400 nM. The lower concentrations of Tat increased reverse transcription efficiency by facilitating multiple rounds of DNA synthesis, and this increase was either not seen or reduced when Tat proteins with multiply-mutated cysteine or basic domains were used. Tat-enhanced reverse transcription occurred in a RNA-independent manner, and required formation of a Tat-RT complex. Pull-down and immunoprecipitation experiments confirmed that Tat could interact with the RT p51 subunit, and mammalian two-hybrid experiments showed interaction between Tat and both the p51 and p66 subunits. Together these results provide evidence that Tat can stimulate reverse transcription through an interaction with RT.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Modelling the fission yeast cell cycle.

The molecular networks regulating basic physiological processes in a cell can be converted into mathematical equations (eg differential equations) and solved by a computer. The division cycle of eukaryotic cells is an important example of such a control system, and fission yeast is an excellent test organism for the computational modelling approach. The mathematical model is tested by simulating wild-type cells and many known cell cycle mutants. This paper describes an example where this approach is useful in understanding multiple rounds of DNA synthesis (endoreplication) in fission yeast cells that lack the main (B-type) mitotic cyclin, Cdc13. It is proposed that the key physiological variable driving progression through the cell cycle during balanced growth and division is the mass/DNA ratio, rather than the mass/nucleus ratio.

Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells.

In mammalian cells, cyclin E-CDK2 complexes are activated in the late G1 phase of the cell cycle and are believed to have an essential role in promoting S-phase entry. We have targeted the murine genes CCNE1 and CCNE2, encoding cyclins E1 and E2. Whereas single knockout mice were viable, double knockout embryos died around midgestation. Strikingly, however, these embryos showed no overt defects in cell proliferation. Instead, we observed developmental phenotypes consistent with placental dysfunction. Mutant placentas had an overall normal structure, but the nuclei of trophoblast giant cells, which normally undergo endoreplication and reach elevated ploidies, showed a marked reduction in DNA content. We derived trophoblast stem cells from double knockout E3.5 blastocysts. These cells retained the ability to differentiate into giant cells in vitro, but were unable to undergo multiple rounds of DNA synthesis, demonstrating that the lack of endoreplication was a cell-autonomous defect. Thus, during embryonic development, the needs for E-type cyclins can be overcome in mitotic cycles but not in endoreplicating cells.

Loss of p19ARF enhances the defects of Mdm2 overexpression in the mammary gland.

The protein encoded by the murine double minute 2 (Mdm2) gene inactivates the function of the tumor suppressor p53. The targeted expression of the mdm2 transgene (BLGmdm2) to the mammary epithelium disrupts the cell cycle, causing multiple rounds of DNA synthesis without proper cell division and consequently poor mammary gland development. These phenotypes in the mammary epithelia of the transgenic mice are not dependent on either p53 or the transcription factor E2F1, as mice null for these genes carrying the BLGmdm2 transgene exhibit similar defects to mice carrying the BLGmdm2 transgene alone. p19ARF, an alternative splice product of the INK4a/ARF locus, has been shown to interact directly with MDM2. Therefore, BLGmdm2 transgenic mice null for p19ARF were created to gain insight into the mechanism by which mdm2 overexpression disrupts the cell cycle. The BLGmdm2 phenotype in the absence of p19ARF was worse than BLGmdm2 mice and visible as early as day 15 of pregnancy. By day 5 of lactation the phenotype was very pronounced. Histological analysis of the mammary gland showed a decrease in ductal branching, smaller and fewer lobuloalveolar structures, and a decrease in luminal secretions. Multinucleated and enlarged cells were present due to continued replication in the absence of cytokinesis. Thus, the absence of p19ARF in this in vivo system enhanced the defect caused by mdm2 overexpression.

Fusions between Epstein-Barr Viral Nuclear Antigen-1 of Epstein-Barr Virus and the Large T-antigen of Simian Virus 40 Replicate Their Cognate Origins

Epstein-Barr viral nuclear antigen-1 (EBNA-1) is required for the stable replication of plasmids that contain oriP, the origin of DNA synthesis used during the latent phase of the Epstein-Barr virus life cycle. EBNA-1 acts post-synthetically through unknown mechanisms to facilitate the continued synthesis of oriP plasmids in ensuing S phases. In contrast to viral replicons such as that of SV40, DNA synthesis of oriP is restricted to a single round during each cell cycle. Large T-antigen of SV40 is a DNA helicase and activates the synthesis of SV40 DNA by recruiting cellular proteins required for DNA synthesis to the origin of SV40. Using fusion proteins of EBNA-1 and large T-antigen, we tested whether tethering large T-antigen to oriP is sufficient to initiate multiple rounds of DNA synthesis from oriP during each cell cycle. We report here that, although these fusion proteins retain the biological activities of both EBNA-1 and large T-antigen, their constituent proteins do not confer the properties of one on the other. Thus, it is not possible to subvert the cellular controls that restrict DNA synthesis from oriP to a single round per cell cycle. These results also provide insights into architectural constraints at oriP and at the SV40 ori.

Replication of R6K γ origin in vitro: discrete start sites for DNA synthesis dependent on π and its copy-up variants

The regulation of the plasmid R6K γ origin (γ ori) is accomplished through the ability of the π protein to act as an initiator and inhibitor of replication. Hyperactive variants of this protein, called copy-up π, allow four to tenfold increases of γ ori plasmid DNA in vivo. The higher activity of copy-up π variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that can be used by wt π (π35.0) only under certain conditions. We have compared the replication activities of wt π35.0and copy-up π mutants in vitro, and analyzed the replication products. It is shown that copy-up variants are several-fold more active than wt π35.0in replication. This appears to be due to enhanced specific replication activity of copy-up mutants rather than elevated fractions of protein proficient in DNA binding. Furthermore, biochemical complementation revealed that π200 (copy-up) is dominant over wt π35.0. The elevated activity of copy-up π is not caused by an increased rate of replisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt π35.0. Moreover, only one round of semiconservative, unidirectional replication occurred in all the samples analyzed indicating that copy-up π proteins do not initiate multiple rounds of DNA synthesis. Rather, a larger fraction of DNA template replicates in the presence of copy-up π as determined by electron microscopy. Two clusters of discrete DNA synthesis start sites are mapped by primer extension near the stability (stb) locus of the γ ori. We show that the start sites are the same in the presence of wt π35.0or copy-up proteins. This comparative analysis suggests that wt π35.0and copy-up variants utilize fundamentally similar mechanism(s) of replication priming.

Activities of SV40 T antigen necessary for the induction of tetraploid DNA content in permissive CV-1 cells

To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV-1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with > G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full-length T antigen alone was responsible for the tetraploid DNA content. The studies show that full-length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing > G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1-259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binding. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis.

Activities of SV40 T antigen necessary for the induction of tetraploid DNA content in permissive CV‐1 cells

To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV-1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with >G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full-length T antigen alone was responsible for the tetraploid DNA content. The studies show that full-length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing >G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1–259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binidng. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis. Cytometry 31:251–259, 1998. © 1998 Wiley-Liss, Inc.

Induction of tetraploid DNA content by mutant simian virus 40 T antigen that has reduced complex formation with p53.

The 402 mutants (DE, DH, DN) of simian virus (SV) 40 form reduced levels of p53-T antigen complexes or no complexes in lytically infected cells (CV-1 cells) relative to wild-type (wt) virus when assayed by immunoprecipitation. When CV-1 cells were infected with the 402 mutants, the cells were induced into multiple rounds of DNA synthesis without mitosis, resulting in a large population of cells with > G2 (tetraploid) DNA content similar to wt virus. The levels of T antigen and p53 per cell that were determined by flow cytometry were similar to wt lytically infected cells, with the levels of T antigen increasing as the infection proceeded. The p53 increased as the levels of T antigen increased, similar to a wt infection. These studies demonstrate that, in lytically infected cells with reduced p53-T antigen complex formation, the cells are induced into multiple rounds of DNA synthesis.

Bovine papillomavirus E1 protein can, by itself, efficiently drive multiple rounds of DNA synthesis in vitro.

Bovine papillomavirus E1 protein was found to be as efficient as the simian virus 40 large T antigen in initiating DNA synthesis in a cell-free system derived from COS1 cells. Multiple rounds of DNA synthesis occur, initiated at the bovine papillomavirus type 1 origin. Therefore, E1 functions in vitro as a lytic virus initiator.

A p53-dependent mouse spindle checkpoint.

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.

Expression of exogenous c‐myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures

Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c-myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super-induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle-related genes if incubated with c-myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with anti-MYC antisera were found to migrate between 55-67 KDa in SDS-PAGE; only the 64-67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.

15] Mapping adducts of DNA structural probes using transcription and primer extension approaches

This chapter presents protocols for the use of both DNA and RNA polymerases to detect adducts. DNA polymerases are used to extend an annealed primer, an approach applied to the analysis of altered DNA structures. Recently, a procedure was developed for using RNA polymerases from phages to map adducts by transcribing modified DNA. The chapter refers to the RNA polymerase approach as transcription and the DNA polymerase approach as primer extension. The principal requirement for the transcription approach is that an appropriate promoter should exist near the sequence to be analyzed. Commonly used promoters are those derived from SP6, T3, and T7 phages. The transcription termination procedure has been successfully tested using SP6, T3, and T7 RNA polymerases, each of which has a high specificity for its cognate promoter. The use of SP6 and T7 RNA polymerases, in separate reactions for each modified sample in a pGEM vector, allows analysis of each strand of the inserted DNA. Many of the generalizations made about transcription also apply to primer extension. The main differences are that the copying enzymes used are different, and have somewhat different sensitivities to the presence of adducts, and that an annealed primer is required for primer extension. Because primers can be directed at any segment of DNA, the analysis of modified DNA is no longer limited to a region contained between two phage promoters. Some genomic sequences can readily be examined with a highly radioactive primer or by multiple rounds of DNA synthesis.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.