Biomedical subjects

R Lurz

Publications and source records attributed to R Lurz.

At least 55 records · Page 3Linked to original sources

PubMed1997

DnaA protein binding to individual DnaA boxes in the Escherichia coli replication origin, oriC.

The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by band-shift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory dnaA box.

Bacterial Proteins↗

PubMed1997

Enhancement of ColE1-type replication promoted by the Gp alpha initiator protein of bacteriophage P4.

Gp alpha, the phage P4 specific replication protein, increases in vitro replication of pCN51, a pBR322 based replicon, by a factor of two. This effect is dependent on DNA polymerase I and requires transcription by host RNA polymerase. Electron microscopic analysis of replicating intermediates indicates that pCN51 replication occurred from the same origin and with the same directionality in the presence and in the absence of Gp alpha. These results reveal that Gp alpha can influence the replication of an heterologous replicon and show that this effect occurs in ColE1-type replicons without altering the normal pattern of initiation. Further analysis of replicating intermediates shows an increase in the average size of the ColE1-type replication 'bubble' obtained in the presence of Gp alpha. It is proposed that Gp alpha interacts with the ColE1 replisome complex at an early replication stage.

Colicins↗

PubMed1997

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

Amino Acid Sequence↗

PubMed1997

The replisome organizer (G38P) of Bacillus subtilis bacteriophage SPP1 forms specialized nucleoprotein complexes with two discrete distant regions of the SPP1 genome.

Initiation of Bacillus subtilis bacteriophage SPP1 DNA replication requires the products of genes 38, 39 and 40 (G38P, G39P and G40P). G38P specifically binds two discrete regions, which are 32.1 kb apart in a linear map of the SPP1 genome. One of these target sites, which maps at the left end of the phage genome, within gene 38, was shown to function as an origin of replication and was therefore termed left origin (oriL). The other site, which lies within a non-coding segment in the late transcribed region on the right end of the genome, was termed oriR. Both sites contain two types of repeated elements (termed Box AB and A + T-rich region). The K(app) for the G38P-oriL DNA and G38P-oriR DNA complexes was estimated to be 1 nM and 4 nM, respectively. G38P binds to the distant oriL and oriR sites cooperatively. DNase I footprinting experiments showed protection by G38P in Box AB, but not in the A + T-rich region. Electron microscopy analysis showed that G38P forms a higher-order nucleoprotein structure with the SPP1 oriL and oriR sites through protein-protein interaction. G38P binding at its cognate sites does not seem to modify the length of the DNA, but to bend it. These results suggest that G38P forms a nucleoprotein complex on the regions where the SPP1 replication origins were previously predicted.

Bacillus Phages↗

PubMed1997

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted. We have assigned genes 7, 8, 9, 11, 12, 13, 14 and 15 to these orfs by correlating genetic and immunological data with DNA and protein sequence information. G7P was identified as a minor structural component of proheads and heads, G11P as the scaffold protein, G12P and G15P as head minor proteins and G13P as the coat protein. Characterization of intermediates in head assembly, which accumulate during infection with mutants deficient in DNA packaging or in morphogenetic genes, allowed the definition of the head assembly pathway. No proteolytic processing of any of the head components was detected. Removal of G11P by mutation leads to the accumulation of prohead-related structures and aberrant particles which are similar to the assemblies formed by purified G13P in the absence of other phage-encoded proteins. The native molecular masses of G11P and G13P are about 350 kDa and larger than 5000 kDa, respectively (predicted molecular masses 23.4 kDa and 35.3 kDa, respectively). G13P, upon denaturation and renaturation, assembles from protomers into some prohead-related structures. The organization of the DNA packaging and head genes of SPP1 resembles the organization of genes in the analogous regions of phage lambda and P22.

Amino Acid Sequence↗

PubMed1997

Bacillus subtilis 168 RecR protein-DNA complexes visualized as looped structures.

The Bacillus subtilis 168 RecR protein bound to duplex DNA in the presence of ATP and divalent cations (Mg2+ and Zn2+) was visualized by electron microscopy as a nearly spherical particle. A RecR homomultimer is frequently located at the intersection of two duplex DNA strands in an interwound DNA molecule, generating DNA loops of variable length. Two individual DNA molecules bound to the same protein are seen at a very low frequency, if at all. The association of RecR with the intersection of two duplex DNA strands is more often seen in supercoiled than with relaxed or linear DNA. The RecR protein displays a slight but significant preference for negatively supercoiled over linear DNA. The minimum substrate size for RecR protein is about 150 bp in length. A possible mechanism for RecR function in DNA repair is discussed.

Bacillus subtilis↗

PubMed1997

Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA).

Helicases are ATP-driven enzymes essential for DNA unwinding. The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 A, and a central channel. Below pH 6, the molecules aggregate into tubular structures. The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 A, beta = 95.2 degrees, and diffract to 3.5 A resolution using rotating-anode Cu Kalpha radiation. Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V(m) = 3.06 A Da(-1), equivalent to a solvent content of 60%. A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2 degrees to the c axis. The two hexamers are oriented head-to-head with point-group symmetry D(6).

Journal Article↗

PubMed1997

High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.).

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.

DNA, Mitochondrial↗

PubMed1997

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.

Adenosine Triphosphatases↗

PubMed1996

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from. A continuous decrease in the number of molecules within each class derived from successive encapsidation rounds indicates that there are several packaging series which end after each headful packaging cycle. The frequency of molecules in each packaging class follows the distribution expected for a sequential mechanism initiated unidirectionally at a defined position in the genome (pac). The heterogeneity of the DNA fragment sizes within each class reveals an imprecision in headful cleavage of approximately 2.5 kb (5.6% of the genome size). The number of encapsidation events in a packaging series (processivity) was observed to increase with time during the infection process. DNA ejection through the tail can be induced in vitro by a variety of mild denaturing conditions. The first DNA extremity to exit the virion is invariably the same that was observed to be bound to the tail, implying that the viral chromosome is ejected with a specific polarity to penetrate the host. In mature virions a short segment of this chromosome end (55 to 67 bp equivalent to 187 to 288 A) is fixed to the tail area proximal to the head (connector). Upon ejection this extremity is the first to move along the tail tube to exit from the virion through the region where the tail spike was attached.

Bacillus Phages↗

PubMed1996

The ColE1 unidirectional origin acts as a polar replication fork pausing site.

Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.

Bacteriocin Plasmids↗

PubMed1996

Electron microscopic investigation of mitochondrial DNA from Chenopodium album (L.).

DNA molecules from mitochondria of whole plants and a suspension culture of Chenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0. 3-183 kb represented 23-26% of all mtDNA molecules in the preparations from the suspension culture and 13-15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rolling-circle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.

Artifacts↗

PubMed1996

A NusG-like protein from Thermotoga maritima binds to DNA and RNA.

The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.

Amino Acid Sequence↗

PubMed1996

Rolling-circle replication of mitochondrial DNA in the higher plant Chenopodium album (L.).

The mitochondrial genomes of higher plants are larger and more complex than those of all other groups of organisms. We have studied the in vivo replication of chromosomal and plasmid mitochondrial DNAs prepared from a suspension culture and whole plants of the dicotyledonous higher plant Chenopodium album (L.). Electron microscopic studies revealed sigma-shaped, linear, and open circular molecules (subgenomic circles) of variable size as well as a minicircular plasmid of 1.3 kb (mp1). The distribution of single-stranded mitochondrial DNA in the sigma structures and the detection of entirely single-stranded molecules indicate a rolling-circle type of replication of plasmid mp1 and subgenomic circles. About half of the sigma-like molecules had tails exceeding the lengths of the corresponding circle, suggesting the formation of concatemers. Two replication origins (nicking sites) could be identified on mpl by electron microscopy and by a new approach based on the mapping of restriction fragments representing the identical 5' ends of the tails of sigma-like molecules. These data provide, for the first time, evidence for a rolling-circle mode of replication in the mitochondria of higher plants.

DNA Replication↗

PubMed1995

The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region.

Initiation of SPP1 DNA packaging requires the gene 1 and gene 2 products (G1P and G2P), which are different subunits of the terminase enzyme. G1P specifically recognizes the phage packaging initiation region (pac). The apparent equilibrium constant for the G1P-pac-DNA complex was estimated to be 9 nM. DNase I footprinting experiments reveal that the pac region can be subdivided into three discrete sites (pacL, pacC and pacR). G1P binds co-operatively to the non-adjacent pacL and pacR sites. Several G1P protomers bind to the target sequences which map close to the pac cleavage site (pacC site), but do not overlap with it. G1P interacts in a different fashion with the encapsidated (pacR site) and with the non-encapsidated (pacL site) end of the phage genome. G1P interaction with the intrinsically bent pacL DNA occurs only on one face of the DNA double helix. G1P binding to the pacL and in the pacR region results in a DNA loop. Electron microscopy of purified G1P shows that the protein is an oligomer in solution. G1P binding to the core region of the pacL site could facilitate the formation of a higher-order nucleoprotein structure. This specialized complex would allow the pac DNA to form a loop between binding sites brought together by interaction with G1P. The results presented here suggest that G1P could provide a tool to discriminate the first encapsidated end, which contains pacR, from the non-encapsidated pacL end.

Bacillus Phages↗

PubMed1995

The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon.

Bacteriophage P1↗

PubMed1995

Transcription of repA, the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator.

Transcription of the repA gene of the Pseudomonas plasmid pPS10 is initiated from a sigma 70 type promoter located 81 bp upstream from the repA gene, extends through the repA gene and the adjacent open reading frame, and ends 1114 nucleotides downstream. The repA promoter is repressed by interactions of the RepA protein with a region of 44 bp that extends from the -10 box of the promoter to the dnaA box of the origin of replication. The core of the repA operator region is formed by two in-phase invertedly repeated sequences of 8 bp, S1 and S2, that flank the -35 box of the promoter, and that share homology with the internal sequences of the iterons present in the origin of replication. RepA enters at the operator region first by protein-DNA interactions and subsequently by protein-protein interactions. These sequential interactions lead to the formation of high, medium and low-mobility electrophoretic complexes. Formation of the high-order complexes seems to be important for an efficient repression of the promoter. Interactions of RepA with the repA promoter region (repPO) occur more efficiently than with the origin of replication.

Bacterial Proteins↗

PubMed1995

Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.

Amino Acid Sequence↗