DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork

Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis

A Pleiotrophin C-terminus peptide induces anti-cancer effects through RPTPβ/ζ

<p>Abstract</p> <p>Background</p> <p>Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor.</p> <p>Results</p> <p>Our results show that P(122-131) inhibits <it>in vitro </it>adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPβ/ζ. In addition, P(122-131) inhibits angiogenesis <it>in vivo</it>, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk¹/₂. Finally, P(122-131) not only interacts with RPTPβ/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPβ/ζ expression with the RNAi technology.</p> <p>Conclusions</p> <p>In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPβ/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.</p

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems

The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

<p>Abstract</p> <p>Background</p> <p>Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit <it>in vitro </it>and <it>in vivo </it>the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.</p> <p>Methods</p> <p>A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. <it>In vitro</it>, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. <it>In vivo</it>, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. <it>In vivo </it>anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.</p> <p>Results</p> <p>Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. <it>In vitro</it>, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an <it>in vivo </it>Matrigel™ plug assay in mice</p> <p>Conclusions</p> <p>Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on <it>in vitro </it>and <it>in vivo </it>growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). <it>In vivo</it>, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.</p

Effets anti-tumoraux de peptides issus de la structure du facteur de croissance HARP via les récepteurs ALK et RPTPb

HARP est un facteur de croissance jouant un rôle-clé dans la progression et dans l invasion tumorale. C est une molécule multifonctionnelle possédant des activités mitogène, transformante et angiogène. L activité de HARP est mediée par deux types de récepteurs transmembranaires; RPTPb et ALK. La structure de HARP est constituée de deux domaines homologues aux TSR motifs et de deux régions basiques aux extrémités N- et C-terminaux. Les travaux précédents de notre équipe ont montré l implication de la région C-terminal de HARP dans ses activités dépendantes de ALK. Le but de mon travail de thèse a été d approfondir ces recherches et plus particulièrement sur les relations structure-fonction de HARP. Dans ce dessein, nous avons synthétisé deux peptides dérivant de l extrémité C-terminal de HARP: les peptides P111-124 et P122-131. Le peptide P111-124 a inhibé les activités mitogène et transformante de HARP dans un modèle de cancer de prostate humain, entrant en compétition avec la fixation de HARP sur son récepteur ALK. Ce peptide inhibe également la croissance tumorale de ces cellules dans les souris athymiques et des études pharmacocinétiques ont confirmé la présence du peptide dans la tumeur et sa bio distribution efficace après l injection intraveineuse. Le peptide P122-131 a inhibé la migration cellulaire, l adhésion et l angiogénèse induite par HARP via RPTPb. Des études de biosensor, utilisant les peptides correspondant aux domaines C-terminal et TSR de HARP nous ont permis de mieux comprendre le mécanisme d action de HARP via les voies de signalisation HARP/ALK/héparine/RPTPb. Enfin, des études portant sur la recherche d autres partenaires ont mis en évidence que HARP entre en interaction avec de nombreuses molécules telles que FGF2, FGF-BP, VEGF, NPN-1, HGF/SF, S100A4 et OPN. En conclusion, ce travail ouvre de nouvelles perspectives portant sur l étude du mécanisme moléculaire de HARP et sur l utilisation potentielle de deux peptides ciblant ALK et RPTPb dans le traitement du cancer.HARP is a heparin-binding growth factor, which plays a key role in tumor growth and invasion. It is a multifunctional polypeptide with mitogenic, transforming and angiogenic activities. HARP mediates its diverse functions through its main transmembrane receptors: RPTPb and ALK. Structurally, HARP contains two TSR homologous domains and two basic clusters in its N and C-termini. Previous studies from our laboratory have reported that the ALK-dependent biological activities of HARP were related to its C-terminal region including the amino acid residues 111-136. The aim of this thesis was to further elucidate the structure-function relationship of HARP. In this aim, we designed two synthetic peptides derived from the Cterminal region. P111-124 inhibited the mitogenic and transforming activities of HARP in a prostatic cancer cell model, by specifically competing with HARP for binding to ALK. The peptide inhibited the tumor growth of these cells in nude mice and the in vivo pharmacokinetic studies indicated the efficient targeting of the peptide to the tumor. P122-131 inhibited HARPinduced cell migration, adhesion and in vivo angiogenesis, by binding to RPTPb. The biosensor studies using the C-terminal and TSR peptides allowed us to better understand the mechanisms that underpin the signaling features of HARP/ALK/heparin/RPTPb. Finally, the binding assays with biosensor revealed the interaction of HARP with a subset of proteins including FGF2, FGFBP, VEGF, NPN-1, HGF/SF, S100A4 and OPN. In conclusion, this thesis yielded new insights in mechanism of action of HARP. The discovery of new molecular partners for HARP implies new avenues for regulatory interplay of the protein. The thesis also highlights the potential of the two anti-tumoral peptides targeting ALK and RPTPb, as therapeutical agents against cancer.PARIS12-Bib. électronique (940280011) / SudocSudocFranceF

The three-component helicase/primase complex of herpes simplex virus-1

Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase–primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase–primase complex for development of alternative anti-viral treatments

Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy and Enzymatic Assays

The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ