Biological consequences of a reduction in the non-target DNA scanning capacity of a DNA repair enzyme

Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.     

Crystallization and preliminary diffraction studies of Erythrina trypsin inhibitor

Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.     

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.     

Enhancement of macrophage IL-1 production by Leishmania major infection in vitro and its inhibition by IFN-gamma

Peritoneal cells from highly susceptible BALB/c mice were infected with Leishmania major and cultured for various times in vitro. The culture supernatants contained significant levels of IL-1 which were consistently higher than those in the cell cultures stimulated with an optimal concentration of LPS. This finding extends to a macrophage cell line, P388D1, and peritoneal exudate cells stimulated with starch in vivo. However, the level of IL-1 produced was significantly reduced when the cells were preincubated with a lymphokine preparation (supernatant of Con A-stimulated rat spleen cells). The level of IL-1 produced seems to be directly correlated with the degree of parasitization of the macrophages. A similar and dose-dependent reduction in IL-1 production by infected macrophages could also be obtained when the cells were preincubated with IFN-gamma. This finding is in direct contrast to that of visceral leishmaniasis in which peritoneal macrophages from BALB/c mice infected with Leishmania donovani not only fail to produce IL-1 but also lose the capacity to produce IL-1. This apparent discrepancy is discussed in terms of a possible difference in the induction of cell-mediated immunity between the two leishmanial diseases.     

The addition of exogenous gangliosides to cultured human cells results in the cell type-specific expression of novel surface antigens by a biosynthetic process

After the observation that human mAb 32-27M reacts only with melanoma and astrocytoma cells cultured in the presence of fetal bovine serum, a novel pathway for the uptake of exogenous gangliosides, their further biosynthesis, and expression at the cell surface as novel Ag has been elucidated. The addition of fetal bovine serum to melanoma and astrocytoma cells growing in synthetic medium (insulin-transferrin-selenium) resulted in reactivity with Ab32-27M. As antibody 32-27M detects N-glycolylneuraminic acid (NeuGc)-containing gangliosides, the effect of adding a number of different gangliosides to melanoma and astrocytoma cells cultured in the synthetic medium was studied. Only the addition of NeuGc-GM3 resulted in the development of Ab32-27M reactivity. The identity of the antigenic structures developed after addition of fetal bovine serum or NeuGc-GM3 was determined by analysis of the gangliosides from both samples. The major component detected in melanoma cell lines was shown to be N-acetylneuraminic acid-NeuGc-GD3. Another, slower moving component, present in some melanomas and in astrocytomas may be N-acetylneuraminic acid-NeuGc-GD2. The cell type specificity for these processes can be most readily explained by postulating that all cells can take up exogenous gangliosides but only melanoma and astrocytoma cells have sufficiently high levels of GM3 alpha 2----8-sialyltransferase for the conversion of added NeuGc-GM3 to disialogangliosides to be effective. These results demonstrate a novel pathway for exogenous glycolipid processing that can lead to novel Ag expression but may also play a role in normal glycolipid metabolism and function.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.     

Consequences of molecular engineering enhanced DNA binding in a DNA repair enzyme.

Facilitated one-dimensional diffusion is a general mechanism utilized by several DNA-interactive proteins as they search for their target sites within large domains of nontarget DNA. T4 endonuclease V is a protein which scans DNA in a nonspecifically bound state and processively incises DNA at ultraviolet (UV)-induced pyrimidine dimer sites. An electrostatic contribution to this mechanism of target location has been established. Previous studies indicate that a decrease in the affinity of endonuclease V for nontarget DNA results in a decreased ability to scan DNA and a concomitant decrease in the ability to enhance UV survival in repair-deficient Escherichia coli. This study was designed to question the contrasting effect of an increase in the affinity of endonuclease V for nontarget DNA. With this as a goal, a gradient of increasingly basic amino acid content was created along a proposed endonuclease V-nontarget DNA interface. This incremental increase in positive charge correlated with the stepwise enhancement of nontarget DNA binding, yet inversely correlated with enhanced UV survival in repair-deficient E. coli. Further analysis suggests that the observed reduction in UV survival is consistent with the hypothesis that enhanced nontarget DNA affinity results in reduced pyrimidine dimer-specific recognition and/or binding. The net effect is a reduction in the efficiency of pyrimidine dimer incision.     

Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

A modular domain of NifU,a nitrogen fixation cluster protein,is highly conserved in evolution

HnifU, a gene exhibiting similarity tonifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins fromHaemophilus influenzae andSaccharomyces cerevisiae, respectively, and 40–44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63–77%) than to the diazatrophic NifU proteins (40–48%). Further, the NifU-like proteins of non-nitrogenfixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans andH. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins. Correspondence to: C.-C. Liew