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Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.

According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilageto-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.osteoblast ontogeny | chondrocyte lineage | bone repair I n vertebrates, the endochondral bones of the axial and appendicular skeleton (1) develop from mesenchymal progenitors that form condensations in the approximate shape of the future skeletal elements. These progenitors differentiate into chondrocytes, which proliferate, mature, and undergo hypertrophy, forming an avascular cartilaginous template surrounded by a perichondrium. The first osteoblasts differentiate from mesenchymal precursors in the perichondrium and produce a bone collar, which will become the future cortical bone (1). Blood vessels then invade through the bone collar into the hypertrophic cartilage, bringing in osteoblast progenitors from the perichondrium (2), which lay down bone matrix to form the primary ossification center (POC); the cartilage matrix is degraded; and the proximal and distal growth plates, comprising layers of differentiating chondrocytes and spongy/trabecular bone (the primary spongiosa), form (2). Thereafter, linear bone growth continues by endochondral ossification mediated by the growth plate, whereas osteoblasts in the perichondrium form cortical bone on the outer circumference.Chondrocytes and osteoblasts are regarded as separate lineages in development, being derived from common mesenchymal progenitors that express the transcription factors sex determining region Y (SRY)-box 9 (Sox9) and runt related transcription factor 2 (Runx2) (1). Lineage determination toward the chondrocyte or osteoblast fate is controlled by the relative expression of Sox9 and Runx2 (3-5) (Fig. 1A). Sox9 controls chondrocyte proliferation and their progression into hypertrophy (6). Collagen X is the most specific marker of hypertrophic chondrocytes (HCs), the Col10a1 gene being expressed only in prehypertrophic and hypertrophic chondrocytes in the growth plate (7). By contrast, Runx2 is essential for specifying the osteoblast lineage an...

Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

For efficient DNA replication of papillomaviruses, only two viral-encoded proteins, El and E2, are required. Other proteins and factors are provided by the host cell. E2 is an enhancer ofboth transcription and replication and is known to help El bind cooperatively to the origin of DNA replication. El is sufficient for replication in extracts prepared from permissive ceils, but the activity is enhanced by E2. Here we show that purified El can act as an ATP-dependent DNA helicase. To measure this activity, we have used strand displacement, unwinding of topologically constrained DNA, denaturation of duplex fragments, and electron microscopy. The ability of El to unwind circular DNA is found to be independent of origin-specific viral DNA sequences under a variety of experimental conditions. In unfractionated cellular extracts, El-dependent viral DNA replication is origin-dependent, but at elevated El concentrations, replication can occur on nonorigin-containing DNA templates. This conversion from an origin-dependent replication system to a nonspecific initiator system is discussed in the context of the current understanding of the initiation of chromosomal DNA replication.Helicases are critical enzymes in the semiconservative replication of DNA (1). Although some polymerases can melt duplex DNA and progressively catalyze strand displacement ahead of the growing polynucleotide chain, this melting is usually inefficient and requires the aid of a helicase. Helicases also work in conjunction with proteins having a strong affinity for single-stranded (ss) DNA, which stabilize the melted duplex as the helicase catalyzes processive unwinding. Often a helicase is brought to the site on DNA where replication initiates by interaction with specific DNA binding proteins that preassemble at the origin of DNA replication. For Escherichia coli or its A phages, the DnaB helicase is efficiently loaded onto the respective origin sites by DnaA and DnaC (2) or the A-encoded 0 and P proteins (3). In eukaryotes, little is known as to how cellular helicases may become associated with replication complexes, although, for some of the well-studied animal viruses such as the polyomaviruses and the herpesviruses, viral-encoded helicases are also equipped to be site-specific DNA binding proteins that can recognize the start site for replication. Other auxiliary factors may, therefore, not be required for loading. The simian virus 40 large tumor antigen is such an originrecognizing helicase (4) that initiates replication by unwinding the origin site as the complex cellular polymerizing machinery assembles (5, 6). Herpes simplex virus 1 (HSV-1) encodes two proteins that can catalyze DNA strand displacement on helicase substrates, and one of these proteins binds specifically to the duplex HSV-1 origin of replication, although extensive duplex unwinding has not been detected (7, 8).We have described an in vitro replication system for bovine papilloma virus 1 (BPV-1) DNA (9, 10) that may provideThe publication costs of this article were d...

Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV.

Chemotaxing cells, such as Dictyostelium and mammalian neutrophils, sense shallow chemoattractant gradients and respond with highly polarized changes in cell morphology and motility. Uniform chemoattractant stimulation induces the transient translocations of several downstream signaling components, including phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In contrast, static spatial chemoattractant gradients elicit the persistent, amplified localization of these molecules. We have proposed a model in which the response to chemoattractant is regulated by a balance of a local excitation and a global inhibition, both of which are controlled by receptor occupancy. This model can account for both the transient and spatial responses to chemoattractants, but alone does not amplify the external gradient. In this article, we develop a model in which parallel local excitation, global inhibition mechanisms control the membrane binding of PI3K and PTEN. Together, the action of these enzymes induces an amplified PI(3,4,5)P3 response that agrees quantitatively with experimentally obtained plekstrin homology-green fluorescent protein distributions in latrunculin-treated cells. We compare the model's performance with that of several mutants in which one or both of the enzymes are disrupted. The model accounts for the observed response to multiple, simultaneous chemoattractant cues and can recreate the cellular response to combinations of temporal and spatial stimuli. Finally, we use the model to predict the response of a cell where only a fraction is stimulated by a saturating dose of chemoattractant.