Plasmid Substrates Research Articles

Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding

The Enterococcus faecalis prg and pcf genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis in vitro and blocked pCF10 transfer in vivo. As shown with affinity pulldown assays, PrgJ and the K471E mutant protein interacted with the substrate receptor PcfC and with relaxase PcfG and accessory factor PcfF, which together form the relaxosome at the oriT sequence to initiate plasmid processing. The purified PrgJ and K471E proteins also bound single- and double-stranded DNA substrates without sequence specificity in vitro, and both PrgJ derivatives bound pCF10 in vivo by a mechanism dependent on an intact oriT sequence and cosynthesis of PcfC, PcfF, and PcfG, as shown by a formaldehyde-cross-linking assay. Our findings support a model in which the PcfC receptor coordinates with the PrgJ ATPase to drive early steps of pCF10 processing/transfer: (i) PcfC first binds the pCF10 relaxosome through contacts with PcfF, PcfG, and DNA; (ii) PcfC delivers the plasmid substrate to PrgJ; and (iii) PrgJ catalyzes substrate transfer to the membrane translocase. Substrate engagement with a VirB4-like subunit has not been previously described; consequently, our studies point to a novel function for these signature T4SS ATPases in mediating early steps of type IV secretion.

Interplay between mismatch repair and chromatin assembly

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

Assembly and in vitro Functional Analysis of Zinc Finger Nuclease Specific to the 3′ Untranslated Region of Chicken Ovalbumin Gene

Synthetic zinc finger nucleases (ZFNs) are useful for the improvement of site directed integration of foreign gene into vertebrate chromosomes. To facilitate site-directed integration of foreign genes into the 3′-untranslated region of the chicken ovalbumin gene, we have constructed ZFN expression vectors using Zinc Finger Consortium Vector Kits and tested the functionality of these ZFN constructs. Coding sequences for 6 zinc fingers were assembled following the modular assembly method. The zinc finger assembly was fused to two FokI catalytic domains. Various configurations of linker regions between domains were tested for their influence on enzymatic activity, using plasmid substrate containing the target sequence. Results indicated that ZFN with an elongated linker between two nuclease domains had a high catalytic activity.

Secalonic acid D as a novel DNA topoisomerase I inhibitor from marine lichen-derived fungus Gliocladium sp. T31

Context: DNA topoisomerase I (topo I) is an essential enzyme which regulates the conformational changes in DNA topology by cleaving and rejoining DNA strands during normal cell growth. The inhibitors of topo I represent a major class of anticancer drugs. In our projects to isolate new anticancer agents from marine-derived fungi, secalonic acid D (SAD) with inhibitory activity on topo I was isolated from the fermentation broth of marine lichen-derived fungus Gliocladium sp. T31, which was collected from marine sediments in South Pole.Objective: The inhibitory activity of SAD on topo I was investigated for the first time.Materials and methods: The inhibitory effect of SAD on topo I was determined via in vitro supercoil relaxation assays and electrophoretic mobility shift assay (EMSA) using plasmid substrate, pBR322.Results: SAD displays a considerable inhibition on topo I in a dose-dependent manner with the minimum inhibitory concentration (MIC) of 0.4 µM. Unlike the prototypic DNA topo I poison camptothecin (CPT), SAD inhibits the binding of topo I to DNA but does not induce the formation of topo I-DNA covalent complexes.Discussion and conclusion: SAD is an excellent topo I inhibitor and thus a significantly potential anticancer candidate.

8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1

The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of transcription by 8-oxo-G fully depends on 8-oxoguanine DNA glycosylase (OGG1) and, at the same time, does not require the localization of the lesion in the transcribed DNA strand. We propose that the interruption of transcription is induced by base excision repair intermediates and, therefore, could be a common consequence of various DNA base modifications. Concordantly, the non-blocking DNA modification uracil was also found to inhibit transcription, but in an OGG1-independent manner.

Uracil excision repair in Mycobacterium tuberculosis cell-free extracts

Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria.

Critical Amino Acid Residues Within the ɸC31 Integrase DNA-Binding Domain Affect Recombination Activities in Mammalian Cells

The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.

A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads

In this paper we extend the application area of the label-free structure-sensitive electrochemical DNA sensing with mercury-based electrodes which is for the first time used, in combination with immunoprecipitation at magnetic beads (MB), for the probing of DNA interactions with tumor suppressor protein p53. The technique relies on capture of the p53–DNA complexes at MB via anti-p53 antibodies, followed by salt-induced dissociation of linear DNA from the complex and its voltammetric detection. Competitive binding of p53 to various plasmid DNA substrates, including lin or scDNAs with or without a specific target site, can easily be followed by ex situ electrochemical analysis of DNA recovered from the immunoprecipitated complexes. Compared to gel electrophoresis which is usually applied to analyze different plasmid DNA forms and their complexes with proteins, the electrochemical detection is faster and allows simpler quantitation of DNA containing free ends at submicrogram levels. We demonstrate applicability of the proposed technique to monitor different DNA-binding activities of wild type and mutant p53 proteins.

TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends

DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA-PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA-PK end binding and activation step and (2) DNA-PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDDeltaN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (DeltaL(k) = -4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDDeltaN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.

An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA

A rigorous understanding of the biological function of superhelical tension in cellular DNA requires the development of new tools and model systems for study. To this end, an ethidium bromide[#x02013]free method has been developed to prepare large quantities of either negatively or positively super-coiled plasmid DNA. The method is based upon the known effects of ionic strength on the direction of binding of DNA to an archaeal histone, rHMfB, with low and high salt concentrations leading to positive and negative DNA supercoiling, respectively. In addition to fully optimized conditions for large-scale (>500 microg) supercoiling reactions, the method is advantageous in that it avoids the use of mutagenic ethidium bromide, is applicable to chemically modified plasmid DNA substrates, and produces both positively and negatively supercoiled DNA using a single set of reagents.

Mechanical Constraints on Hin Subunit Rotation Imposed by the Fis/Enhancer System and DNA Supercoiling during Site-Specific Recombination

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence elements. First, a selectable marker is PCR-amplified with synthetic primers attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary restriction site absent in the substrate plasmid. The resulting cassette is co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli cells. Following isolation of recombinant plasmids, linear nonselectable DNA replaces the cassette and introduces the desired mutation(s) in a second Red/ET recombination step. Upon selective digestion of parental plasmids and retransformation, a 38% mutation efficiency was achieved using a synthetic 97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19 (2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp).

Error‐Free Repair of CAG/CTG Hairpins via Endonucleolytic Incisions in Human Nuclear Extracts

Expansion of trinucleotide repeats (TNRs) is associated with progression of several human neurological diseases including Huntington's disease. The proposed mechanism of TNR expansion involves replication‐associated slippage, leading to the formation of TNR hairpins. Thus, factors preventing or removing TNR hairpins are predicted to inhibit disease progression and promote TNR stability. To determine how human cells process TNR hairpins, circular plasmid DNA substrates containing a (CAG)25 or (CTG)25 hairpin were constructed and analyzed for repair by human nuclear extracts. We show that human cells catalyze error‐free repair of (CAG)25 and (CTG)25 hairpins in a nick‐directed PCNA‐dependent manner. The repair is initiated by incisions specifically targeting the repeat sequences in the nicked strand, followed by repair DNA synthesis using the continuous strand as a template, thereby ensuring TNR stability. Depletion of PCNA blocks repair prior to TNR incision. However, distinct incision products were observed in reactions containing TNR hairpins with different strand locations and secondary structures, indicating utilization of distinct endonucleases in different hairpin repair. Our findings on TNR hairpin repair will provide significant insight into the mechanisms of TNR expansion and the etiology of human diseases caused by TNR expansions.

Collateral Damage from Antigen Receptor Gene Diversification

Chromosomal translocations that juxtapose antigen receptor genes and oncogenes are frequently associated with lymphoid malignancies. In this issue, Robbiani et al. (2008) show that activation-induced deaminase (AID), an enzyme involved in antigen receptor gene diversification, generates DNA double-strand breaks (DSBs) in oncogenes, and Tsai et al. (2008) propose that AID and the recombinase-activating gene (RAG) endonuclease may collaborate to generate off-target DSBs.

Molecular analysis of recombinase-mediated cassette exchange reactions catalyzed by integrase of coliphage HK022

The integrase (Int) protein of coliphage HK022 can catalyze in Escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. Atomic force microscopy images have revealed that in the protein–DNA complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. This observation, together with the elucidation of intermediate co-integrates between the two circular plasmids, suggest that a sequential mechanism of the RMCE reaction is possible.

Activation of 12/23-RSS-Dependent RAG Cleavage by hSWI/SNF Complex in the Absence of Transcription

Maintenance of genomic integrity during antigen receptor gene rearrangements requires (1) regulated access of the V(D)J recombinase to specific loci and (2) generation of double-strand DNA breaks only after recognition of a pair of matched recombination signal sequences (RSSs). Here we recapitulate both key aspects of regulated recombinase accessibility in a cell-free system using plasmid substrates assembled into chromatin. We show that recruitment of the SWI/SNF chromatin-remodeling complex to both RSSs increases coupled cleavage by RAG1 and RAG2 proteins. SWI/SNF functions by altering local chromatin structure in the absence of RNA polymerase II-dependent transcription or histone modifications. These observations demonstrate a direct role for cis-sequence-regulated local chromatin remodeling in RAG1/2-dependent initiation of V(D)J recombination.

Mathematical Model of Growth and Heterologous Hantavirus Protein Production of the Recombinant Yeast Saccharomyces cerevisiae

AbstractA segregated mathematical model was developed for the analysis and interpretation of cultivation data of growth of the recombinant yeast Saccharomyces cerevisiae on multiple substrates (glucose, maltose, pyruvate, ethanol, acetate, and galactose). The model accounts for substrate consumption, plasmid stability, and production level of a model protein, a modified nucleocapsid protein of the Puumala virus. Recombinant nucleocapsid proteins from different Hantaviruses have previously been demonstrated as suitable antigens for diagnostics as well as for sero‐epidemiological studies. The model is based on a system of 10 nonlinear ordinary differential equations and accounts for the influence of various factors, e.g., selective pressure for enhancing plasmid stability by formaldehyde or the toxic effects of the intracellular accumulation of the heterologous protein on cell growth and product yield. The model allows the growth of two populations of cells to be simulated: plasmid‐bearing and plasmid‐free yeast cells, which have lost the plasmid during cultivation. Based on the model, sensitivity studies in respect to parameter changes were performed. These enabled, for example, the evaluation of the impact of an increase in the initial concentration of nutrients and growth factors (e.g., vitamins, microelements, etc.) on the biomass yield and the heterologous protein production level. As expected, the productivity of the heterologous protein in S. cerevisiae is closely correlated with plasmid stability. The 25 free model parameters, including the yield coefficients for different growth stages and dynamic constants, were estimated by nonlinear techniques, and the model was validated against a data set not used for parameter estimation. The simulation results were found to be in good agreement with the experimental data.

Engineering Variants of the I-SceI Homing Endonuclease with Strand-specific and Site-specific DNA-nicking Activity

The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3′-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstrate that although wild-type I-SceI cleaves either the top or bottom DNA strand first to generate two nicked DNA intermediates, the enzyme has a preference for cleaving the bottom strand. The kinetics data are consistent with a parallel sequential reaction mechanism. Substitution of two pseudo-symmetric residues, Lys122 and Lys223, markedly reduces top and bottom-strand cleavage, respectively, to generate enzymes with significant strand- and sequence-specific nicking activity. The two active sites are partially interdependent, since alterations to one site affect the second. The kinetics analysis is consistent with X-ray crystal structures of I-SceI/DNA complexes that reveal a role for the lysines in establishing important solvent networks that include nucleophilic water molecules thought to attack the scissile phosphodiester bonds.