The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells.

Collectins are a family of C-type lectins that have collagen-like sequences and carbohydrate recognition domains (CRD). They are involved in host defense through their ability to bind to carbohydrate antigens of microorganisms. The scavenger receptors type A and MARCO are classical type scavenger receptors that have internal collagen-like domains. Here we describe a new scavenger receptor that is a membrane-type collectin from placenta (collectin placenta 1 (CL-P1)), which has a typical collectin collagen-like domain and a CRD. The cDNA has an insert of about 2.2 kilobases coding for a protein containing 742 amino acid residues. The deduced amino acid sequence shows that CL-P1 is a type II membrane protein, has a coiled-coil region, a collagen-like domain, and a CRD. It resembles type A scavenger receptors because the scavenger receptor cysteine-rich domain is replaced by a CRD. Northern analyses, reverse transcription-polymerase chain reaction, and immunohistochemistry show that CL-P1 is expressed in vascular endothelial cells but not in macrophages. By immunoblotting and flow cytometry CL-P1 appears to be a membrane glycoprotein of about 140 kDa in human umbilical vein or arterial endothelial cells, placental membrane extracts, and CL-P1 transfected Chinese hamster ovary cells. We found that CL-P1 can bind and phagocytose not only bacteria (Escherichia coli and Staphylococcus aureus) but also yeast (Saccharomyces cerevisiae). Furthermore, it reacts with oxidized low density lipoprotein (OxLDL) but not with acetylated LDL (AcLDL). These binding activities are inhibited by polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate) and OxLDL but not by polycationic ligands (polyadenylic acid or polycytidylic acid), LDL, or AcLDL. These results indicate that CL-P1 might play important roles in host defenses that are different from those of soluble collectins in innate immunity.

Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions.

Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escherichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical characterization of the gene product expressed in E. coli. The Tth ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and made the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, whose ATPase activity is strongly enhanced by supercoiled DNA but only weakly enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was efficiently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB hydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migration of a synthetic Holliday junction at 60 degrees C in an ATP-dependent manner. The protein, as judged by its ATPase activity, required ATP for thermostability. Since a RuvA protein has not yet been identified in T. thermophilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E. coli RuvA greatly enhanced the ability of Tth RuvB to hydrolyze ATP in the presence of DNA and to promote branch migration of a synthetic Holliday junction at 37 degrees C. These results indicate the conservation of the RuvA-RuvB interaction in different bacterial species, and suggest the existence of a ruvA homolog in T. thermophilus. Although GTP and dGTP were efficiently hydrolyzed by Tth RuvB, these nucleoside triphosphates could not be utilized for branch migration in vitro, implying that the conformational change in RuvB brought about by ATP hydrolysis, which is necessary for driving the Holliday junction branch migration, cannot be accomplished by the hydrolysis of these nucleoside triphosphates.

Identification of functional domains of Bordetella dermonecrotizing toxin.

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin stress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rho. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutants. Our analyses of the nucleotide and amino acid sequences revealed that the start codon of the DNT gene is a GTG triplet located 39 bp upstream of the reported putative initiation ATG codon; consequently, DNT contains an additional 13 amino acids at its N-terminal end. All of the N-terminally truncated mutants were found to modify Rho. The shortest fragment of DNT possessing the Rho modification activity consists of amino acids from Ile1176 to the C-terminal end. This fragment overlaps the region homologous to Escherichia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the highly conserved region between CNFs and DNT eliminated the activity, indicating that this domain is the catalytic center of DNT. The N-terminal fragment (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynucleation in MC3T3-E1 cells when simultaneously added with the holotoxin, suggesting competitive inhibition in the receptor-binding or internalizing step. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facilitate analysis of the overall action of the toxin on the mammalian target cells.

Molecular cloning of a novel human collectin from liver (CL-L1).

Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains. These proteins can bind to carbohydrate antigens of microorganisms and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway, and opsonization by collectin receptors. Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1 (collectin liver 1)) that has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot, Western blot, and reverse transcription-polymerase chain reaction analyses showed that CL-L1 is present mainly in liver as a cytosolic protein and at low levels in placenta. More sensitive analyses by reverse transcription-polymerase chain reactions showed that most tissues (except skeletal muscle) have CL-L1 mRNA. Zoo-blot analysis indicated that CL-L1 is limited to mammals and birds. A chromosomal localization study indicated that the CL-L1 gene localizes to chromosome 8q23-q24.1, different from chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in Escherichia coli affirmed that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan columns. Analysis of the phylogenetic tree of CL-L1 and other collectins indicated that CL-L1 belongs to a fourth subfamily of collectins following the mannan-binding protein, surfactant protein A, and surfactant protein D subfamilies including bovine conglutinin and collectin-43 (CL-43). These findings indicate that CL-L1 may be involved in different biological functions.

R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase.

Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells. The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase. A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA. Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication. This mitochondrial R-loop is resolved by RecG in a dose-dependent manner. Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG. A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop. This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase. The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG.

Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning.

BACKGROUND: All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically. RESULTS: After a low UV dose (5 J/m2), both delta ruvAB and delta ruvC mutant cells became filamentous, with their chromosomes aggregated in the central region. This corresponded to an increase in nonmigrating DNA on pulsed field gel electrophoresis of the XbaI digested chromosome. Upon further incubation, they produced a large number of anucleoid cells of normal size. A recA mutation, but not a recB mutation, suppressed these phenotypes of the ruv mutants. The ruv polA12(Ts) double mutants were inviable at the nonpermissive temperature and mimicked the morphological phenotypes of the UV irradiated ruv mutants. CONCLUSION: ruvA, B and C mutations block chromosome partitioning in UV irradiated cells because the abortive homologous recombination covalently links chromosomes together. There is a recBCD independent pathway for the recA dependent formation of recombination intermediates. An Ruv-mediated resolution of recombination intermediates is required for the repair of strand breaks produced in UV irradiated cells and in the polA mutant cells.

ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase.

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.