Biomedical subjects

W Messer

Publications and source records attributed to W Messer.

At least 37 records · Page 2Linked to original sources

PubMed1999

The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA.

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo-oligomerization of DnaA via its N-terminus (amino acid residues 1-86) is also essential for initiation. Results from solid-phase protein-binding assays indicate that residues 1-86 (or 1-77) of DnaA are necessary and sufficient for self interaction. Using a 'one-hybrid-system' we found that the DnaA N-terminus can functionally replace the dimerization domain of coliphage lambda cl repressor: a lambdacl-DnaA chimeric protein inhibits lambda plasmid replication as efficiently as lambdacI repressor. DnaA derivatives with deletions in the N-terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N-terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N-termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross-talk with the nucleotide-binding domain III or the DNA-binding domain IV. We propose that E. coli DnaA is composed of largely independent domains - or modules - each contributing a partial, though essential, function to the proper functioning of the 'holoprotein'.

Amino Acid Sequence↗

PubMed1999

Interactions of the Streptomyces lividans initiator protein DnaA with its target.

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.

Amino Acid Sequence↗

PubMed1998

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium.

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinus dnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.

Amino Acid Sequence↗

PubMed1998

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Bacterial Proteins↗

PubMed1998

High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli.

The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC. Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests. Except for oriC, these fragments contain only one or two consensus DnaA binding sites, DnaA boxes. The distribution of these high-affinity DnaA boxes on the chromosome is random.

Bacterial Proteins↗

PubMed1998

Transcriptional analysis and mutation of a dnaA-like gene in Synechocystis sp. strain PCC 6803.

Transcription of the dnaA gene of the cyanobacterium Synechocystis sp. strain PCC 6803 is light dependent and yields a monocistronic mRNA, as determined by Northern analysis. Surprisingly, mutants with inactivated dnaA were viable. In batch cultures under standard conditions, the mutants grew like the wild type and did not show an aberrant phenotype. We conclude that, unlike the situation in other bacteria, dnaA of Synechocystis sp. cannot have an essential function, such as initiation of DNA replication.

Bacterial Proteins↗

PubMed1997

Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein.

The initial steps in the formation of the initiation complex at oriC of Bacillus subtilis were analyzed with special emphasis on the exchangeability of B. subtilis DnaA protein by DnaA of Escherichia coli. The DNA binding domain of B. subtilis DnaA protein was localized in the 93 C-terminal amino acids. Formation of the "initial complex", as analyzed by electron microscopy, was indistinguishable with B. subtilis DnaA protein or with E. coli DnaA. Similarly, both proteins were able to form loops by interaction of DnaA proteins bound to the DnaA box regions upstream and downstream of the dnaA gene in B. subtilis oriC. The region of local unwinding in the "open complex" was precisely defined. It is located at one side of a region of helical instability, a DNA unwinding element (DUE). Unwinding in oriC could only be catalyzed by the homologous DnaA protein.

Amino Acid Sequence↗

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

From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA.

The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus sequence: 5'-TTA/TTNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique. (i) dU at the positions of T2, T4, T7' or T9' completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T3' on the complementary strand of teh box 5'-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position. In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.

Bacterial Proteins↗

PubMed1997

DnaA protein stimulates polA gene expression in Escherichia coli.

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprinting experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.

Bacterial Proteins↗

PubMed1997

DnaA initiator--also a transcription factor.

The replication-initiator protein DnaA is ubiquitous in the eubacterial world. It binds to an asymmetric 9 bp consensus DNA sequence, the DnaA box. Besides its primary function as an initiator, it acts as a transcription factor that represses or activates several genes, or terminates transcription, depending on the location and arrangement of DnaA boxes.

Bacterial Proteins↗

PubMed1997

Purification and characterization of the Streptomyces lividans initiator protein DnaA.

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.

Adenosine Triphosphatases↗

PubMed1997

Prediction of the structure of the replication initiator protein DnaA.

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an "open twisted alpha/beta structure," containing five alpha-helices alternating with five beta-strands. In our proposed structural model the interior of the core is formed by a parallel beta-sheet, whereas the alpha-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first beta-strand. The N-terminal domain (80 aa) is composed of two alpha-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a beta-strand and an additional alpha-helix. The N-terminal domain and the alpha/beta core region of DnaA are connected by a variable loop (45-70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly alpha-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal alpha-helices could interact with each other, forming an internal "coiled coil" and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts.

Amino Acid Sequence↗

PubMed1996

A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli.

We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo.

Bacterial Proteins↗

PubMed1995

Interaction of the initiator protein DnaA of Escherichia coli with its DNA target.

Equilibrium and kinetic rate constants were determined for the binding of the initiator protein DnaA of Escherichia coli to its binding site, the non-palindromic 9-bp DnaA box, using gel retardation techniques. The dissociation constant for specific binding was between 1 and 50 nM for individual DnaA boxes on 21-bp double-stranded oligonucleotides. Only DnaA boxes of the sequence TT(A/T)TNCACA resulted in specific fragment retention. Both the 9-bp consensus sequence and flanking sequences determined the binding efficiency. One DnaA monomer was found to bind to a DnaA box and to induce a bend of about 40 degrees.

Bacterial Proteins↗

PubMed1995

The DNA binding domain of the initiator protein DnaA.

The 94 C-terminal amino acids of the initiator protein DnaA of Escherichia coli are required and sufficient for specific binding to the cognate DNA binding site. The binding domain contains two potential amphipathic alpha-helices and a third alpha-helix. It represents a new DNA binding motif so far not found in other DNA binding proteins. Temperature-sensitive mutations in the binding motif, dnaA204, dnaA205 and dnaA211, abolish DNA binding. In the solid-phase DNA binding assay, applicable to other DNA binding proteins, fusions of domains of DnaA protein to beta-galactosidase are reacted with biotinylated anti-beta-galactosidase antibody. These are coupled to streptavidin-coated magnetic beads. The DNA binding domain is able to selectively remove the DNA target (oriC) from the liquid phase. Alternatively, the DNA binding domain is fused to a peptide containing a target sequence which is naturally biotinylated in vivo in E.coli. This fusion protein can be coupled directly to streptavidin-coated magnetic beads. Homologies between DnaA protein and transcription factors of the NtrC family are discussed.

Amino Acid Sequence↗

PubMed1995

Genetic structure of the dnaA region of the cyanobacterium Synechocystis sp. strain PCC6803.

We have cloned and sequenced the dnaA region of Synechocystis sp. strain PCC6803, a bacterium with a light-dependent cell cycle. The dnaA gene product, DnaA, is the central factor for replication initiation in bacteria. The deduced amino acid sequence of the protein encoded by the cyanobacterial dnaA gene is 45% identical to DnaA of Bacillus subtilis and fits very well into the homology pattern of the known eubacterial DnaA proteins. The genetic environment of the Synechocystis sp. strain PCC6803 dnaA gene is completely different from the one in other eubacteria. An open reading frame of unknown function, orf134, was detected upstream of dnaA. The purT gene homolog encoding the glycinamide ribonucleotide transformylase T starts about 200 bp away from this open reading frame in the opposite direction. Downstream of the dnaA gene we detected the start of the psbDC operon, which codes for the photosystem II reaction center proteins D2 and CP43 that are involved in the positioning of chlorophyll a.

Acyltransferases↗

PubMed1994

Functions of histone-like proteins in the initiation of DNA replication at oriC of Escherichia coli.

Using methidiumpropyl-EDTA (MPE) footprinting we found one specific binding site for FIS protein in the E coli replication origin, oriC. We mutagenized the binding sites for FIS and IHF in oriC and analyzed the effect of the mutations on protein binding and oriC function. The replication efficiency of oriC plasmids paralleled the ability of the mutated DNA fragments to bind IHF or FIS. We conclude that these histone-like proteins function in cis in the initiation of DNA replication at oriC.

Base Sequence↗