A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-beta-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to beta-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

Multiple functional domains are involved in tomosyn regulation of exocytosis

Tomosyn is a cytoplasmic protein that was shown to bind to Syntaxin1 and SNAP-25 through an R-SNARE domain, forming a complex that is almost identical in structure to the neuronal SNARE complex. Tomosyn inhibits exocytosis in various cell types and these effects were attributed to direct competition between tomosyn's SNARE domain and Synaptobrevin/VAMP. In the present study, we investigated the contribution of different domains of tomosyn to its activity. We show that a tomosyn mutant that lacks the entire SNARE domain is a potent inhibitor of vesicle priming, similar to the full-length tomosyn. The SNARE domain of tomosyn failed to inhibit exocytosis, indicating that this domain is not required for the inhibition. In contrast, over-expression of a N-terminally truncated mutant did not lead to inhibition of exocytosis although this mutant still bound to Syntaxin. Our results indicate that tomosyn can inhibit exocytosis independently of its SNARE interaction with Syntaxin and that the integrity of the WD40-domain is crucial for tomosyn's inhibitory function. Furthermore, we demonstrate that the entire N-terminal region of tomosyn, the WD40-repeats and the linker, is required for tomosyn's inhibitory effect.     

Multiple domains of occludin are involved in the regulation of paracellular permeability

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.     

Inhibition of DNA methyltransferase inhibits DNA replication

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.     

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.     

Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.     

Determination of the functional domains involved in nucleolar targeting of nucleolin.

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.     

Distinct domains of paracingulin are involved in its targeting to the actin cytoskeleton and regulation of apical junction assembly

Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.     

DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability.

DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.     

Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.     

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation.     

Centromeres are specialized replication domains in heterochromatin

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.     

Mapping of Eps15 domains involved in its targeting to clathrin-coated pits

Clathrin-coated pit (CCP) formation occurs as a result of the targeting and assembly of cytosolic coat proteins, mainly the plasma membrane clathrin-associated protein complex (AP-2) and clathrin, to the intracellular face of the plasma membrane. In the present study, the mechanisms by which Eps15, an AP-2-binding protein, is targeted to CCPs was analyzed by following the intracellular localization of Eps15 mutants fused to the green fluorescent protein. Our previous results indicated that the N-terminal Eps15 homology (EH) domains are required for CCP targeting. We now show that EH domains are, however, not sufficient for targeting to CCPs. Similarly, neither the central coiled-coil nor the C-terminal AP-2 binding domains were able to address green fluorescent protein to CCPs. Thus, targeting of Eps15 to CCPs likely results from the collaboration between EH domains and another domain of the protein. An Eps15 mutant lacking the coiled-coil domain localized to CCPs showing that Eps15 dimerization is not strictly required. In contrast, Eps15 mutants lacking all AP-2 binding sites showed a dramatic decrease in plasma membrane staining, showing that AP-2 binding sites, together with EH domains, play an important role in targeting Eps15 into CCPs. Finally, the effect of the Eps15 mutants on clathrin-dependent endocytosis was tested by both immunofluorescence and flow cytometry. The results obtained showed that inhibition of transferrin uptake was observed only with mutants able to interfere with CCP assembly.     

The microarchitecture of DNA replication domains

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures—replication bodies (RB), and (2) focal replication sites with no distinct underlying structure—replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase α, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase α, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.     

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA. Modification of both the 70- and 32-kDa subunits, RPA70 and RPA32, respectively, by photoaffinity labeling indicates that RPA can bind the primer-template junction of partial duplex DNAs by interacting with the 3'-end of the primer. The identification of the protein domains modified by the photoreactive 3'-end of the primer showed that domains located in the central part of the RPA32 subunit (amino acids 39-180) and the C-terminal part of the RPA70 subunit (amino acids 432-616) are involved in these interactions.     

Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.