Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

Characterization of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter: nuclear factors that interact with an element shared by three lymphokine genes--those for GM-CSF, interleukin-4 (IL-4), and IL-5.

The region extending from -40 to -54 of the 5'-flanking region of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene shows homology to sequences found in the 5'-flanking regions of other cytokine genes, those encoding interleukin-4 (IL-4), IL-5, and granulocyte colony-stimulating factor (G-CSF). This sequence element is referred to as conserved lymphokine element 0 (CLE0). Saturation mutagenesis of the CLE0 element indicates that in addition to the previously mapped region between -73 and -91 (CLE2+ GC box), the CLE0 element is necessary for induction of the mouse GM-CSF gene by phorbol myristate acetate/Ca ionophore (A23187) stimulation in T cells. The presence of the CLE0 element is necessary to observe stimulation of the transcription activity of the mouse GM-CSF promoter in vitro. Mobility shift assays revealed that this region forms an inducible DNA-protein complex, NF-CLE0, which consists of two complexes of similar mobility, NF-CLE0a and NF-CLE0b. NF-CLE0a and NF-CLE0b recognize the 3' half and 5' half of the CLE0 element, respectively, with an overlapping region recognized by both proteins. The recognition sequence of NF-CLE0a corresponds to the region required for induction by phorbol myristate acetate/A23187, while the recognition sequence of NF-CLE0b contains bases that have inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

Chitinase secreted by Leishmania functions in the sandfly vector.

Leishmania major parasites ingested with host blood by the sandfly Phlebotomus papatasi multiply confined within the peritrophic membrane. This membrane consists of a chitin framework and a protein carbohydrate matrix and it is secreted around the food by the insect midgut. Histological sections of infected flies show lysis of the chitin layer in the anterior region of the peritrophic membrane that permits the essential forward migration of a concentrated mass of parasites. Both the location and the nature of this disintegration are specific to infected flies. At a later stage the parasites concentrate in the cardiac valve region and subsequently this segment of the fore gut loses its cuticular lining. We have found that chitinase and N-acetylglucosaminidase are secreted by cultured L. major promastigotes, but not by sandfly guts. Hence lysis of the chitin layer of the peritrophic membrane could be catalysed by these enzymes of the parasites. Activity of both enzymes was also observed in other trypanosomatids, including L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculata and Trypanosoma lewisi.

Vector compatibility of Phlebotomus papatasi dependent on differentially induced digestion.

Infection with Leishmania tropica, a strain specific to the sandfly Phlebotomus papatasi, was inhibited in sandflies fed on turkey blood. Reduction of the parasite number was correlated with the digestive process. A relatively high DNAase level was induced in the gut of the sandfly by the nucleated turkey erythrocytes. This is the first record of vector-pathogen incompatibility, thus induced, and of differentially triggered digestive processes.

A unique ATP-dependent DNA topoisomerase from trypanosomatids.

Crithidia fasciculata DNA topoisomerase (22) has been purified to near homogeneity from trypanosomatid cell extracts. The purified enzyme catalyzes the reversible interconversion of monomeric duplex DNA circles and catenanes in an ATP dependent reaction. Reversible catenane formation is affected by the ionic strength and is dependent upon the action of a crithidial DNA binding protein, which could be substituted for the polyamine spermidine. Covalently sealed DNA circles are specifically used as substrates for decatenation. Nicking, but not relaxation per se, inhibits network decatenation and has little or no effect on catenane formation. The catalytic properties of this enzyme and its potential role in the prereplication release and post replication reattachment of kDNA minicircles are discussed.

The assembly of kinetoplast DNA.

The unusual structure of the kinetoplast DNA (kDNA) of trypanosomatids requires unique replication mechanisms. Deciphering the mechanisms that regulate the network assembly has been a challenge for many years. A better understanding of these processes was facilitated by recent studies on the fine structure of resting and replicating kDNA networks. In this review, Joseph Shlomai discusses our current view of the structural and mechanistic aspects of the assembly of kinetoplast DNA.

A unique endonuclease from Crithidia fasciculata which recognizes a bend in the DNA helix. Specificity of the cleavage reaction.

The introduction of a single nick in DNA circles by Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) requires the presence of a bent structure in the DNA helix. However, the sequence directing the local bending of the DNA helix is not per se a preferred site for nicking by the enzyme. No extensive sequence specificity is involved in defining the cleavage site for C. fasciculata nicking enzyme in the duplex circular DNA substrate. However, the abundance of A and T residues is significantly high at both the 3' and the 5' termini generated at the nicked site. Nicking of the sequence-directed bent fragment from C. fasciculata kinetoplast DNA minicircles correlates with the periodicity determined by the unique nucleotide distribution in the bent sequence, reflected in its thermodynamic parameters. Occurrence of nicking is best correlated with the predicted minima of the melting temperature and delta G profiles, as well as with A and T dinucleotide sequences at the nicked site, in both the supercoiled and the relaxed sequence-directed bent DNA substrates. The potential role of the bend-dependent nicking reaction in the replication of kinetoplast DNA minicircles is discussed.

Replication of herpes simplex virus DNA after removal of hydroxyurea block from infected cells.

Hydroxyurea (HU) treatment of HSV-infected cells markedly inhibits the synthesis of virus DNA. Only 0-3.6 of the 3H-thymidine label was incorporated into virus DNA in the presence of HU as compared to untreated infected cells. Removal of HU resulted in a renewed synthesis of virus DNA as determined by the gradual increase in the incorporation of 3H-thymidine into HSV DNA. The labelled virus DNA molecules were isolated and chromatographed on benzoylated napthoylated DEAE (BND)-cellulose columns to separate the replicative intermediates that have single-stranded (ss) sequences from the mature double-stranded (ds) DNA genomes. Mature radioactive dsDNA molecules were found to appear at 22 min after removal of HU and gradually increased in amount thereafter. The virus DNA molecules synthesized during the initial 20 min after removal of HU, constitute the replicative intermediates of HSV DNA. It was calculated that the synthesis of HSV DNA proceeds at the rate of about 5 X 10(6) daltons per min.

A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization.

Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.

Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids.

Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms. The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells. Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles. The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence. As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S. cerevisiae.

Kinetoplast DNA minicircles of trypanosomatids encode for a protein product.

The major constituent of the trypanosomal kinetoplast DNA network are several thousand duplex DNA minicircles whose biological function is still unknown. The coding capacity and expression of these DNA minicircles, was studied in the trypanosomatid Crithidia fasciculata. Kinetoplast DNA minicircle fragments inserted into bacterial plasmid vectors were expressed in the bacterial cell. Sera elicited in rabbits, by immunization with the translational products of kinetoplast DNA minicircles in E. coli, reacted specifically with Crithidia fasciculata cellular antigens. It is inferred that kinetoplast DNA minicircles contain long open reading frames of nucleotides which are expressed in the trypanosomatid cell.

Reversible decatenation of kinetoplast DNA by a DNA topoisomerase from trypanosomatids.

DNA topoisomerase activity detected in cell extracts of the trypanosomatid Crithidia fasciculata interlocks kinetoplast DNA duplex minicircles into huge catenane forms resembling the natural kinetoplast DNA networks found in trypanosomes. Catenation of duplex DNA circles is reversible and equilibrium is affected by ionic strength, and by spermidine. The reaction requires magnesium, is ATP dependent and is inhibited by high concentrations of novobiocin. Extensive homology between duplex DNA rings was not required for catenane formation since DNA circles with unrelated sequences could be interlocked into mixed network forms. Covalently sealed catenaned DNA circles are specifically used as substrates for decatenation. No such preference for covalently sealed duplex DNA rings was observed for catenate formation. Its catalytic properties and DNA substrate preference, suggest a potential role for this eukaryotic topoisomerase activity in the replication of kinetoplast DNA.

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

Protein n', a prepriming DNA replication enzyme of Escherichia coli, is a phi X174 DNA-dependent ATPase. Restriction of phi X174 DNA have led to the identification of a 55-nucleotide fragment that carries the protein n' recognition sequence. Molecular hybridization and sequence analysis have located this sequence within the untranslated region between genes F and G, a map location analogous to that of the unique complementary strand origin of phage G4 DNA. Within the 55-nucleotide fragment is a sequence of 44 nucleotides that forms a stable hairpin structure. This duplex may be the signal for protein n' to initiate the prepriming events that led to the start of phi X174 complementary DNA strand replication.

A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase.

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.

A prepriming DNA replication enzyme of Escherichia coli. I. Purification of protein n': a sequence-specific, DNA-dependent ATPase.

Protein n', an enzyme essential for in vitro conversion of single-stranded phiX174 DNA to the duplex replicative form, has been purified about 16,000-fold from Escherichia coli. The enzyme is a single polypeptide chain with a native molecular weight of 76,000; about 70 enzyme molecules are present in an E. coli cell. Nearly homogeneous preparations display an ATPase (dATPase) activity which depends on a unique sequence in the phiX174 DNA. Replicative activity of n' protein and its phiX174 DNA-dependent ATPase activity were present in a constant ratio during the latter stages of purification, upon sedimentation in a glycerol gradient, and during heat inactivation. Further studies of the properties of protein n' are presented in a succeeding paper.

The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata.

Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.