Recombinant expression and characterization of yeast Mrc1, a DNA replication checkpoint mediator

Abstract

In Saccharomyces cerevisiae, Mrc1 (homolog of human Claspin and mediator of replication checkpoint) is not only a part of the replication machine, but also participates in the replication stress response when DNA replication is blocked by hydroxyurea. Since Mrc1 is expressed in a small amount in cells and has many proteins interacting with it as a mediator, it is difficult to obtain Mrc1 with high concentration and purity. This article reports the purification of a stable truncation of Mrc1 and the full length Mrc1. High concentration and high purity of Mrc1 was obtained and the three-dimensional structure of Mrc1 was analyzed, which is a ring with a hole in the center. At the same time, we found that Mrc1 has an interaction with Rad24-RFC a clamp loader in the replication checkpoint, and can form a complex with it, implying that we can assemble large replication checkpoint complexes in vitro. These results initially reveal the ring structure of Mrc1 and its interaction with Rad24-RFC in replication checkpoints in S. cerevisiae.     

Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

Interaction of porcine circovirus type 2 replication with intracellular redox status in vitro

Abstract

Objectives

Redox status influences replication of some viruses but its effect on porcine circovirus type 2 (PCV2), the primary causative agent of the emerging swine disease post-weaning multisystemic wasting syndrome is not known. The interaction of PCV2 replication with intracellular redox status in PK15 cells was examined in this study.

Methods

Intracellular glutathione (GSH) was measured spectrophotometrically by reaction with 5, 5′-dithiobis (2-nitrobenzoic acid). Total superoxide dismutase activity (SOD) was assayed by inhibition of oxyamine oxidation by the xanthine oxidase system. Malondialdehyde (MDA) was assayed spectrophotometrically using the thiobarbituric acid reaction. Both quantification of PCV2 DNA by real-time polymerase chain reaction and indirect immunofluorescence of PCV2-infected cells were used to evaluate the replication of PCV2.

Results

Both GSH and SOD decreased significantly at 48 hours after PCV2 infection, whereas MDA concentration increased significantly after 48 hour post-infection. Furthermore, PCV2 replication in PK15 cells was significantly impaired after the elevation of intracellular GSH through treatment with the antioxidant N-acetyl-l-cysteine (NAC), a precursor in GSH synthesis. In contrast, PCV2 replication in PK15 cells was enhanced after reduction of GSH levels through H₂O₂-mediated oxidation. In addition, NAC treatment blocked the increase of virus replication induced by H₂O₂.

Conclusions

This study suggests that PCV2 infection induces oxidative stress and that intracellular redox status influences PCV2 replication in PK15 cells.     

Synthesis and Biological Application of a New Heterodinucleotide with Both Anti-HSV and Anti-HIV Activity

Abstract

A new antiviral drug with both anti-HSV and anti-HIV activity was synthesized by coupling Acyclovir and the acyclic nucleoside phosphonate (R)PMPA. The heterodinucleotide ACVpPMPA encapsulated into autologous erythrocytes was added to human macrophages providing an effective in vitro protection from HSV-1 and HIV-1 replication.     

Stereospecificity of 6′-C-Neplanocin A Analogues as Inhibitors of S-Adenosylhomocysteine Hydrolase Activity and Human Immunodeficiency Virus Replication†

Abstract

The R- and S-isomers of 6′-C-neplanocin A analogues, which are all known as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were studied for their inhibitory effects on Human Immunodeficiency Virus type 1 (HIV-1) replication and HIV-1 Tat-mediated transactivation. The R-isomers showed much greater activity against AdoHcy hydrolase than the S-isomers. The same differential activity was observed against the HIV-1 replication and the Tat transactivation.

     

A biomimetic platform for studying root-environment interaction

Aims

Microstructure plays an important role in biological systems. Microstructural features are critical in the interaction between two biological organisms, for example, a microorganism and the surface of a plant. However, isolating the structural effect of the interaction from all other parameters is challenging when working directly with the natural system. Replicating microstructure of leaves was recently shown to be a powerful research tool for studying leaf-environment interaction. However, no such tool exists for roots. Roots present a special challenge because of their delicacy (specifically of root hairs) and their 3D structure. We aim at developing such a tool for roots.

Methods

Biomimetics use synthetic systems to mimic the structure of biological systems, enabling the isolation of structural function. Here we present a method which adapts tools from leaf microstructure replication to roots. We introduce new polymers for this replication.

Results

We find that Polyurethane methacrylate (PUMA) with fast UV curing gives a reliable replication of the tomato root surface microstructure. We show that our system is compatible with the pathogenic soilborne bacterium Ralstonia solanacearum.

Conclusions

This newly developed tool may be used to study the effect of microstructure, isolated from all other effects, on the interaction of roots with their environment.

     

Base Composition Skews, Replication Orientation, and Gene Orientation in 12 Prokaryote Genomes

Variation in GC content, GC skew and AT skew along genomic regions was examined at third codon positions in completely sequenced prokaryotes. Eight out of nine eubacteria studied show GC and AT skews that change sign at the origin of replication. The leading strand in DNA replication is G-T rich at codon position 3 in six eubacteria, but C-T rich in two Mycoplasma species. In M. genitalium the AT and GC skews are symmetrical around the origin and terminus of replication, whereas its GC content variation has been shown to have a centre of symmetry elsewhere in the genome. Borrelia burgdorferi and Treponema pallidum show extraordinary extents of base composition skew correlated with direction of DNA replication. Base composition skews measured at third codon positions probably reflect mutational biases, whereas those measured over all bases in a sequence (or at codon positions 1 and 2) can be strongly affected by protein considerations due to the tendency in some bacteria for genes to be transcribed in the same direction that they are replicated. Consequently in some species the direction of skew for total genomic DNA is opposite to that for codon position 3. Received: 2 February 1998 / Accepted: 15 June 1998     

A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.

     

Cyanobacterial multi-copy chromosomes and their replication

ABSTRACT

While the model bacteria Escherichia coli and Bacillus subtilis harbor single chromosomes, which is known as monoploidy, some freshwater cyanobacteria contain multiple chromosome copies per cell throughout their cell cycle, which is known as polyploidy. In the model cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, chromosome copy number (ploidy) is regulated in response to growth phase and environmental factors. In S. elongatus 7942, chromosome replication is asynchronous both among cells and chromosomes. Comparative analysis of S. elongatus 7942 and S. sp. 6803 revealed a variety of DNA replication mechanisms. In this review, the current knowledge of ploidy and DNA replication mechanisms in cyanobacteria is summarized together with information on the features common with plant chloroplasts. It is worth noting that the occurrence of polyploidy and its regulation are correlated with certain cyanobacterial lifestyles and are shared between some cyanobacteria and chloroplasts.     

Synthesis,In Vitro Anti-HIV and Anti-hepatitis B Activities and Pharmacokinetic Properties of Amphiphilic Heterodinucleoside Phosphates Containing ddC and AZT

Abstract

Amphiphilic heterodinucleoside phosphates containing AZT and ddC as antiviral monomer were synthesized according to the hydrogenphosphonate method and evaluated in vitro against HIV. dT-N⁴-pamddC was the most active (IC₅₀ = 40 μM, EC₅₀ = 80 nM) and least toxic (TI = 524) dimer and it exhibited also strong antiviral effects against eight AZT-resistant HIV strains. The ddC-containing heterodimers additionally inhibited HBV replication by 50–80% at 50 μM in Hep G2 2.2.15 cells.     

Deletion analysis of ors12, a centromeric,early activated,mammalian origin of DNA replication

We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized. J. Cell. Biochem. 66:87–97, 1997. © 1997 Wiley-Liss, Inc.     

The three most important things about origins: location,location, location

The reasons why some DNA replication origins fire earlier than others have remained elusive. New work by Gindin et al suggests that the distribution of replication origins, not their timing per se, is the major determinant of the timing of genome replication in human cells.     

Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse

Elevated mistranslation induces a mutator response termed translational stress‐induced mutagenesis (TSM) that is mediated by an unidentified modification of DNA polymerase III. Here we address two questions: (i) does TSM result from direct polymerase corruption, or from an indirect pathway triggered by increased protein turnover? (ii) Why are homologous recombination functions required for the expression of TSM under certain conditions, but not others? We show that replication of bacteriophage T4 in cells expressing the mutA allele of the glyV tRNA gene (Asp→Gly mistranslation), leads to both increased mutagenesis, and to an altered mutational specificity, results that strongly support mistranslational corruption of DNA polymerase. We also show that expression of mutA, which confers a recA‐dependent mutator phenotype, leads to increased lambdoid prophage induction (selectable in vivo expression technology assay), suggesting that replication fork collapse occurs more frequently in mutA cells relative to control cells. No such increase in prophage induction is seen in cells expressing alaV^Glu tRNA (Glu→Ala mistranslation), in which the mutator phenotype is recA‐independent. We propose that replication fork collapse accompanies episodic hypermutagenic replication cycles in mutA cells, requiring homologous recombination functions for fork recovery, and therefore, for mutation recovery. These findings highlight hitherto under‐appreciated links among translation, replication and recombination, and suggest that translational fidelity, which is affected by genetic and environmental signals, is a key modulator of replication fidelity.     

Mode and origin of replication of pSAM2, a conjugative integrating element of Streptomyces ambofaciens

pSAM2 is an 11 kb integrating element from Streptomyces ambofaciens that is capable of replication. It generates single-stranded DNA during replication, and is therefore the first Streptomyces integrating element to be described that may belong to the family of elements, called the ssDNA elements, that replicate by a rolling-circle mechanism. The direction of replication has been identified. The plus origin (ori) of replication and minus origin (M-O) have been located. Streptomyces lividans harbouring replicating pSAM2 also contain numerous small covalently closed circular DNA molecules (scm) derived from pSAM2. These scm contain ori and extend on both sides of the putative nick site. Sequences at the junction points of these scm are heterogeneous but short direct repeats were always found in the vicinity of these junctions.     

Molecular cloning,recombinant expression,and antifungal functional characterization of the lipid transfer protein from Panax ginseng

Objectives

To establish an efficient expression system for a fusion protein GST-pgLTP (Lipid Transfer Protein) and to test its antifungal activity.

Results

The nucleotide sequence of LTP gene was obtained from Panax ginseng using RT-PCR. The ORF of the cDNA is 363 bp, codING for a protein OF 120 amino acids with a calculated MW of 12.09 kDa. The pgLTP gene with a His₆-tag at the C-terminus was cloned into the pGEX-6p1 vector to generate a GST-fusion pgLTP protein construct that was expressed in Escherichia coli Rosetta. Following purification by Ni–NTA, the fusion protein exhibited antifungal activity against five fungi found in ginseng.

Conclusion

The fusion protein GST-pgLTP has activity against a broad spectrum of phytopathogenic fungi, and can potentially be adapted for production to combat fungal diseases that affect P. ginseng.

     

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum

ABSTRACT

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40–50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.