William Firshein

Replication of a Bacillus subtilis oriC plasmid in vitro

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis‐elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi‐conservative and only one round of replication occurred within 15min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2‐dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and unidirectional replication occurred in vitro.

Plasmid replication and partition in Escherichiacoli: is the cell membrane the key?

The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non‐covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.

Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2

The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol. 141, 213-222). They include the origin of replication (oriRK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions oriRK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.

Probable Identification of a Membrane-Associated Repressor of Bacillus subtilis DNA Replication as the E2 Subunit of the Pyruvate Dehydrogenase Complex

Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901-7906, 1999). However, the exact sequence was not as important in B. subtilis as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.

Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).

The biosynthesis of DNA by insects. I. The utilization of deoxycytidine by developing adults of the cecropia silkworm.

Abstract The pathway of deoxycytidine utilization was investigated in developing pupae of Hyalophora cecropia . The following intermediates were identified in a single assay system starting with deoxycytidine: deoxyuridine, deoxyuridine monophosphate, thymidine monophosphate, thymidine diphosphate, and thymidine triphosphate. The existence of this pathway was confirmed in several ways in addition to the detection of the actual intermediates. First, injection of [ 14 C]deoxycytidine or [ 14 C]deoxyuridine into developing pupae resulted in the localization of the label in the thymine moiety of DNA and no significant radioactivity was detected in the cytosine moiety. Second, omission of various components from the complete assay system produced a predictable loss of certain intermediates. Third, the activity of other enzymes which might compete with this pathway were not detected or detected in trace amounts, e.g. , deoxycytidine monophosphate deaminase, deoxycytidine kinase, thymidine synthetase, deoxyuridine monophosphate kinase and deoxycytidine degradative enzymes. The enzyme which converts deoxyuridine monophosphate to deoxythymidine monophosphate (thymidylate synthetase) was not detected in diapausing or injured diapausing pupae, but was present in significant amounts in developing pupae. The injection of [ 14 C]thymidine into developing pupae also resulted in the association of the radioactivity with DNA-thymine, but little or no thymidine kinase activity could be detected in the extracts.

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: Involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.

The DNA/membrane fraction of Pneumococcus contains a DNA replication complex☆

A DNA/membrane fraction extracted from cell suspensions of pneumococci (containing polyadenylic acid (poly(A)) and DNA precursors) with sodium lauroyl sarcosinate (Sarkosyl) contains a DNA-replication complex. Almost all of the nascent DNA, 70% of the nascent RNA and protein and 85% of the nascent phospholipid were detected in this fraction. The initial rate of synthesis calculated from incorporation of precursors in vivo corresponds to the incorporation of 2.66 × 105 nucleotides/min/cell. The entire fraction formed a pellicle at the meniscus of CsCl density gradients. Some of the DNA and other macromolecules could be dissociated upon treatment with nucleases, pronase or phospholipase. The sedimentation velocity of nascent DNA in the complex was smaller (9 to 12 s) than that of bulk DNA (16 to 20 s). The DNA/membrane fraction was capable of synthesizing new DNA in vitro with no external DNA template added when either deoxyribonucleoside triphosphates, deoxyribonucleoside mono-phosphates or ribonuoleoside diphosphates were added together with appropriate cofactors. A procedure designed to purify the complex with respect to in vitro DNA synthesis not only increased the specific activity of triphosphate- and mono-phosphate-initiated DNA synthesis but produced a “minimal” complex containing 9% of the total cell DNA and 8% of the protein. Experiments with puromycin suggested that enzyme activities in the DNA/membrane fraction were not due to nascent polypeptide chains on polysomes, but rather to “complete” enzymes.

The biosynthesis of DNA by insects: III. Ribonucleotide reductase activity and the effect of hydroxyurea during the adult development of the Cecropia silkworm

Ribonucleotide reductase activity was examined in developing adults of the silkmoth Hyalophora cecropia. The experiments demonstrate, in vivo and in vitro, the incorporation of labeled ribonucleotides into DNA and the direct enzymatic reduction of labeled CMP by cell-free homogenates of tissues excised from developing adults. Hydroxyurea completely inhibits incorporation of labeled ribonucleotides into DNA in vivo, but does not affect the basal level of DNA synthesis or ribonucleotide reduction in vitro. Evidence is presented which indicates that hydroxyurea may prevent the allosteric activation of the reductase.

Overexpression of the Hda DnaA-related protein in Escherichia coli inhibits multiplication, affects membrane permeability, and induces the SOS response.

The Hda protein, a recently identified DnaA-related protein from Escherichia coli , is part of the AAA+ ATPase family known to be involved with various aspects of initiation of DNA replication in prokaryotes. We report here that overexpression of this membrane-associated protein inhibits