DNA binding and aggregation by carbon nanoparticles

Significant environmental and health risks due to the increasing applications of engineered nanoparticles in medical and industrial activities have been concerned by many communities. The interactions between nanomaterials and genomes have been poorly studied so far. This study examined interactions of DNA with carbon nanoparticles (CNP) using atomic force microscopy (AFM). We experimentally assessed how CNP affect DNA molecule and bacterial growth of Escherichia coli. We found that CNP were bound to the DNA molecules during the DNA replication in vivo. The results revealed that the interaction of DNA with CNP resulted in DNA molecule binding and aggregation both in vivo and in vitro in a dose-dependent manner, and consequently inhabiting the E. coli growth. While this was a preliminary study, our results showed that this nanoparticle may have a significant impact on genomic activities.     

Mapping of Escherichia coli RNA polymerase binding sites on 2-micrometers DNA from Saccharomyces cerevisiae. Heterogeneity within the inverted duplication and evidence for an eukaryotic invertible DNA sequence.

The 2-μm DNA plasmids from Saccharomyces cerevisiae strain H1 and strain HQ/5C were analyzed by electron microscopy for the presence of Escherichia coli RNA polymerase binding sites. On native 2-μm DNA isolated from strain HQ/5C five RNA polymerase binding sites were detected. One further site was mapped on cloned 2-μm DNA type 23 from S. cerevisiae strain H1. This additional site is located at a distance of 2.15 kilobases from EcoRI site B inside one of the inverted duplication (id) sequences. No such binding site could be detected in the other id sequence of the type 23 molecule, thus indicating that the two id sequences of strain H1 differ in at least one short region. The location of the id sequence carrying the RNA polymerase binding site was analyzed in native 2-μm DNA isolated from strain H1 and found to be present on HindIII fragment 2 and absent from HindIII fragment 5. This indicates that at least a part of the id sequences has a fixed position with respect to the unique S segment and further suggests a site specific recombination mechanism for the inversion of one of the unique segments. As a control for the specificity of RNA polymerase binding, we have mapped binding sites on vectors pBR313 and pBR322. The location of the E. coli RNA polymerase binding sites on 2-μm DNA is discussed in relation to the DNA regions expressed in E. coli minicells.     

Isolation of specific ribosome binding sites from single-stranded DNA.

The ability of Escherichia coli ribosomes to protect small specific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage f1 and φX174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f1 and φX174 DNA which are protected differ in sequence from each other.When E. coli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of φX174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAase, and a number of additional polypyrimidines.Double-stranded DNAs such as φX174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specific regions as those protected in single-stranded φX174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery

Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the χ subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between χ and an EcoSSB/ssDNA complex when compared to that of χ to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of χ, showing that χ interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that χ specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to χ at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III.     

Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein

Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the χ-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.     

The Escherichia coli colibactin resistance protein ClbS is a novel DNA binding protein that protects DNA from nucleolytic degradation

Cells employ specific and nonspecific mechanisms to protect their genome integrity against exogenous and endogenous factors. The clbS gene is part of the polyketide synthase machinery (pks genomic island) encoding colibactin, a genotoxin implicated in promoting colorectal cancer. The pks is found among the Enterobacteriaceae, in particular Escherichia coli strains of the B2 phylogenetic group. Several resistance mechanisms protect toxin producers against toxicity of their products. ClbS, a cyclopropane hydrolase, was shown to confer colibactin resistance by opening its electrophilic cyclopropane ring. Here we report that ClbS sustained viability and enabled growth also of E. coli expressing another genotoxin, the Usp nuclease. The recA::gfp reporter system showed that ClbS protects against Usp induced DNA damage. To elucidate the mechanism of ClbS mediated protection, we studied the DNA binding ability of the ClbS protein. We show that ClbS directly interacts with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), whereas ssDNA seems to be the preferred substrate. Thus, the ClbS DNA-binding characteristics may serve bacteria to protect their genomes against DNA degradation.     

Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD

YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.     

An architectural role of the Escherichia coli chromatin protein FIS in organising DNA

The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.     

Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair.     

pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coli and E. faecalis at levels of 10³ and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

DNA modification and functional delivery into human cells using Escherichia coli DH10B

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ~200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd^– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.     

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli

Summary The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.     

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.     

Evidence for regulation of the NADH peroxidase gene (npr) from Enterococcus faecalis by OxyR

We report that the purified Escherichia coli OxyR protein can bind specifically upstream of the gene encoding NADH peroxidase (npr) from Enterococcus faecalis 10C1, to a site located some 144 bp from the promoter. A 34 kDa protein has been identified in crude extracts of E. faecalis that cross-reacts with polyclonal antisera to purified OxyR from E. coli and a protein(s) present in these extracts retards npr DNA fragments in gel shift assays. Taken together with the results of sequence analyses, these observations suggest that enterococcal npr is regulated by OxyR.