Replication origin mutations affecting binding of pSC101 plasmid-encoded Rep initiator protein.

To investigate the role of binding sites for Rep initiation protein in the replication of pSC101, a series of plasmids was constructed which carried different combinations of mutations in three binding sites within the minimal origin of replication. Mutation of all three sites reduced the affinity of purified Rep protein for the origin by 100-fold, as measured by a competition binding assay. Mutations in individual binding sites prevented binding of Rep protein to the mutant site but not to adjacent wild-type sites. Transformation efficiency, copy number, and stability over 150 generations were measured for each of the mutant plasmids. Unlike other similar plasmids related to pSC101, the Rep binding sites were found not to be equivalent. A mutation in the site RS1, proximal to repeated sequences which serve as DnaB helicase entry sites in oriC, had a severe effect on replication activity. A similar mutation in the distal site RS3 caused a reduction in copy number, but the mutant plasmid was stably maintained despite a broadened distribution of copy number within the population. A mutation in the middle RS2 site had no significant effect on pSC101 replication.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF.

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.     

Replication of mini-F plasmid in vitro promoted by purified E protein

Summary An in vitro system for replication of mini-F plasmid DNA was constructed. This system consists of an ammonium sulfate fraction II (Fuller et al. 1981) from Escherichia coli extract, exogeneously added purified E protein encoded by mini-F plasmid, and mini-F DNA in a closed circular form. Experiments with this system showed that the 217 bp DNA region which contains the A+T rich cluster and the four 19 bp direct repeats responsible for incB incompatibility is essential for mini-F DNA replication.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

Detection of the single-stranded DNA of Streptomyces plasmid pSA1.1 and a binding histone-like protein

Abstract Streptomyces plasmid pSA1.1 accumulated single-stranded DNA as replication intermediates in S. lividans ; therefore, this plasmid was considered to replicate by a rolling-circle mechanism. A DNA-binding protein (pI > 9.7 and about 10 kDa) was purified on a denatured DNA-Cellulose column, then on a native DNA-Cellulose column. The N-terminal amino acid sequence of this protein has a high homology with bacterial histone-like proteins. In the gel retardation assay, this protein bound with the single-stranded DNA of pSA1.1. We propose that this protein may participate in the replication of pSA1.1.     

DNA bending of pSC101 ori induced by Rep,a replication initiator protein and IHF

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.     

Replication of plasmid pSC101 in Escherichia coli K12: requirement for dnaA function.

Summary Protoplasts of soybean and N. glauca were induced to fuse with polyethylene glycol (PEG 1540). Up to 39% of the protoplasts in the treated population were heterokaryocytes. When the heterokaryocytes were isolated and individually cultivated they divided indefinitely and each produced many millions of cells within 2–3 months.The chromosomal behaviour of soybean and N. glauca in the hybrids were not synchronous in the first few cell generations and the chromosomes of N. glauca had a tendency to stick together and break into pieces. However, some of the N. glauca chromosomes were still retained in the somatic hybrids after 6 months of culturing. The chromosomes of the N. glauca were reconstructed in such a way that in the later cell generations, the movement of the N. glauca chromosomes were in synchrony with the soybean chromosomes.NRCC NO. 15668     

The interaction of E. coli IHF protein with its specific binding sites

We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences. Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein. We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site. IHF bends the DNA to which it binds. We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

HU, the major histone-like protein of E. coli, modulates the binding of IHF to oriC.

HU is one of the most abundant DNA binding proteins of bacteria. Unlike IHF, integration host factor of Escherichia coli, with which HU shares many properties, including a strong sequence homology and similar predicted structure, HU seems to bind non-specifically to DNA whereas IHF binds to specific sites. In this work we compare the binding characteristics of HU and IHF to a DNA fragment containing the minimal origin of replication of E. coli (oriC) and we analyse the effect of HU on the binding capacity of IHF to this oriC fragment. We show that HU interacts randomly and non-specifically with oriC as opposed to the specific binding of IHF to this same DNA sequence. In addition, we show that HU can modulate the binding of IHF to its specific oriC site. Depending on the relative concentrations of HU and IHF, HU is able either to activate or to inhibit the binding of IHF to oriC.     

DNA repair in E. coli strains deficient in single-strand DNA binding protein

Summary Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb ^- recA^- and ssb ^- uvr^- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.     

The role of E. coli single-stranded DNA binding protein in DNA metabolism

Single-stranded DNA binding proteins have been known for some time to be crucial in many DNA metabolic reactions in both prokaryotes and eukaryotes. Despite a wealth of studies on these proteins we still do not understand their biochemical mechanism of action. Recent studies of the Escherichia coli single stranded DNA binding protein (SSB) are beginning to provide some insight into how this and similar proteins might function.     

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.     

大肠杆菌TorS/TorR二组分体应答蛋白TorR对DNA复制起始的影响

二组分体作为一种信号转导系统在细菌中普遍存在,能够感知外界环境变化并做出应答。细菌中CckA/CtrA、ArcA/ArcB和PhoP/PhoQ二组分体与DNA复制起始和细胞分裂相关,但目前还未见TorS/TorR二组分体对细胞周期及DNA复制影响的相关报道。大肠杆菌TorS/TorR二组分体能够监测细胞周围氧化三甲胺(Trimethylamine oxide, TMAO)的浓度变化,但其是否影响DNA复制起始呢?文章利用流式细胞仪检测了ΔtorS和ΔtorR突变体菌株的复制式样。结果发现,ΔtorS突变菌株每个细胞复制起始原点数目和倍增时间与野生型细胞一致,而ΔtorR突变菌株每个细胞复制起始原点数目多于野生型细胞,说明复制起始发生时间比野生型细胞早。但是过表达TorR蛋白或者共同表达TorS和TorR蛋白都不能使ΔtorR突变体表型恢复为野生型表型。而在野生型和ΔtorR突变细胞中过表达SufD蛋白能使复制起始提早发生,在ΔtorR和ΔsufD双突变细胞中复制起始延迟。所以,TorR可能通过改变sufD基因的表达来间接影响染色体复制起始。     

Maintenance of plasmid pSC101 in Escherichia coli requires the host primase.

The abilities of three Escherichia coli strains with thermosensitive dnaG alleles to maintain plasmids pSC101 or pBR322 or an RP4 derivative were studied at elevated growth temperatures. Under these conditions, pSC101 segregated from cells to a greater extent than did pBR322. No segregation of the primase-encoding RP4 derivative was observed.     

Characterization of and lipopolysaccharide binding to the E. coli LptC protein dimer

Lipopolysaccharide (LPS, endotoxin) is the major component of the outer leaflet of the outer membrane of Gram‐negative bacteria such as Escherichia coli and Salmonella typhimurium. LPS is a large lipid containing several acyl chains as its hydrophobic base and numerous sugars as its hydrophilic core and O‐antigen domains, and is an essential element of the organisms' natural defenses in adverse environmental conditions. LptC is one of seven members of the lipopolysaccharide transport (Lpt) protein family that functions to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of the bacterium. LptC is anchored to the IM and associated with the IM LptFGB₂ complex. It is hypothesized that LPS binds to LptC at the IM, transfers to LptA to cross the periplasm, and is inserted by LptDE into the outer leaflet of the outer membrane. The studies described here comprehensively characterize and quantitate the binding of LPS to LptC. Site‐directed spin labeling electron paramagnetic resonance spectroscopy was utilized to characterize the LptC dimer in solution and monitor spin label mobility changes at 10 sites across the protein upon addition of exogenous LPS. The results indicate that soluble LptC forms concentration‐independent N‐terminal dimers in solution, LptA binding does not change the conformation of the LptC dimer nor appreciably disrupt the LptC dimer in vitro, and LPS binding affects the entire LptC protein, with the center and C‐terminal regions showing a greater affinity for LPS than the N‐terminal domain, which has similar dissociation constants to LptA.     

The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase.

A protein in extracts of Escherichia coli that specifically binds the stabilizing par sequence of pSC101 was identified as DNA gyrase. The purified enzyme protects par against digestion by DNase I and exonuclease III. Competition assays demonstrate that gyrase has a 40-fold higher affinity for the 100-bp par sequence than for nonspecific DNA and that par is the major gyrase-binding site in pSC101 derivatives used in this and other studies. Within par, AT-rich sequences occur with a pronounced 10-bp periodicity that is shifted by 5 bp from a similar periodicity of GC-rich sequences. As judged by DNase I digestion, the GC sequences are exposed on the outside of the DNA wrapped around gyrase. The data suggest that the site-specificity of DNA gyrase may be partly determined by the bendability of the DNA. A 4-bp deletion that interferes with Par function in vivo also reduces the affinity for gyrase in vitro. However, a deletion of par causes little reduction in superhelical density in vivo. We conclude that DNA gyrase, while involved in the Par function, may not affect plasmid stability through its supercoiling activity or by an influence on DNA replication.