Single-stranded Products Research Articles

FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.

Nucleic Acid Unwinding by Hepatitis C Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.

Enzymatic manipulations of DNA oligonucleotides on microgel: towards development of DNA–microgel bioassays

We demonstrate that DNA oligonucleotides covalently coupled to colloidal microgel can be manipulated by T4 DNA ligase for DNA ligation and by Phi29 DNA polymerase for rolling circle amplification (RCA). We also show that the long single-stranded RCA product can generate intensive fluorescence upon hybridization with complementary fluorescent DNA probe. We believe DNA-microgel conjugates can be explored for the development of DNA based bioassays and biosensors.

DNA Unwinding by Escherichia coli DNA Helicase I (TraI) Provides Evidence for a Processive Monomeric Molecular Motor

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

Principles of micro-RNA production and maturation

Micro-RNAs (miRNAs) are a class of approximately 22-nucleotide non-coding RNAs expressed in multicellular organisms. They are first transcribed in a similar manner to pre-mRNAs. The transcripts then go through a series of processing steps, including endonucleolytic cleavage, nuclear export and a strand selection procedure, to yield the single-stranded mature miRNA products. The transcription and processing of miRNAs determines the abundance and the sequence of mature miRNAs and has important implications for the function of miRNAs.

Surface Plasmon Resonance for Detection of Genetically Modified Organisms in the Food Supply

A review is presented demonstrating that biospecific interaction analysis, using surface plasmon resonance (SPR) and biosensor technologies is a simple, rapid, and automatable approach to detect genetically modified organisms (GMOs). Using SPR, we were able to monitor in real-time the hybridization between oligonucleotide or polymerase chain reaction (PCR)-generated probes and target single-stranded PCR products obtained by using as substrates DNA isolated from normal or transgenic soybean and maize. This procedure allows a one-step, nonradioactive detection of GMOs. PCR-generated probes are far more efficient in detecting GMOs than are oligodeoxyribonucleotide probes. This is expected to be a very important parameter, because information on low percentage of GMOs is of great value. Determination of the ability of SPR-based analysis to quantify GMOs should be considered a major research field for future studies, especially for the analyses of food supplies.

Polymorphisms in pulmonary innate immune system genes of swine

Modern pigs are unusually susceptible to severe infectious pneumonia. We hypothesize that single nucleotide polymorphisms (SNPs) in innate immune system genes impair resistance to infection or promote injurious inflammatory responses in swine. The objective of this study was to characterize SNPs that code for amino acid substitutions in porcine surfactant protein-A and -D (SP-A, SP-D), complement component C3d (C3d), and Toll-like receptor 2 (TLR-2). For each gene, RT-PCR products that cover the entire coding region were subjected to single-strand conformational polymorphism (SSCP) analysis. RT-PCR was performed on total RNA extracted from the lungs of randomly chosen diseased pigs submitted to the Animal Health Laboratory, University of Guelph, for diagnostic investigation. Pigs were screened for SNPs by comparing silver-stained banding patterns of digested, single-stranded RT-PCR products separated by SSCP gel electrophoresis. Several different banding patterns were observed in digests of SP-A, SP-D and TLR-2, whereas C3d digests appeared uniform. Sequence comparisons of RT-PCR products representing different banding patterns revealed three coding SNPs in the C-type lectin domain of SP-A. The C-type lectin domain of SP-A has an important role in binding microbial surface carbohydrates and these results suggest that pigs may differ in their innate ability to recognize and defend against some respiratory pathogens. Supported by OMAF, Ontario Pork, and the Ontario Veterinary College

Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes

Linking emulsion PCR (LE-PCR) enables formation of minichromosomes preserving phase information of two polymorphic loci, hence the haplotype. Emulsion PCR confines two amplicons of two linked polymorphic sites on a single template molecule to one aqueous-phase droplet. Linking PCR uses biotinylated, overlapping linking primers to connect these amplicons in the droplet. After LE-PCR, unlinked amplicons are removed on streptavidin-coated magnetic beads and single-stranded runoff products are capped by primer extension. Quantitative ASPCR can then be used to ascertain the haplotypes of the two polymorphic loci on the minichromosomes. Using LE-PCR, we determined the human paraoxonase-1 [PON1] molecular haplotypes at three loci (−909g>c, L55M, Q192R) in women who were compound heterozygotes for −909g>c/L55M (n = 89), −909g>c/Q192R (n = 77) and L55M/Q192R (n = 68). We observed a strong association between PON1 substrate specificity (paraoxon/phenylacetate substrate activity ratios) and −909g>c/Q192R haplotype. We have demonstrated here a powerful molecular haplotyping technology that can be applied in population studies.

Native Replication Intermediates of the Yeast 20 S RNA Virus Have a Single-stranded RNA Backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide.

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.

Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis.

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)-PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80-250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFDelta508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

Low-density addressable array for the detection and typing of the human papillomavirus

We have developed a low-density DNA array for the detection and typing of human papillomavirus (HPV) DNA. The gene chemistry strategy involves using a combination of the polymerase chain reaction (PCR) with the consensus oligonucleotide primers MY09/MY11 followed by a ligase detection reaction (LDR). Fluorochrome-labeled HPV-specific primers are joined to a common primer modified with a unique anchoring sequence called a zip code on its 3′ end. The result is a series of 60-70 base pair and single-stranded ligation products that are then hybridized to their respective zip code complements affixed to glass slide based arrays. Nine separate zip codes were assigned, one for each HPV type (6,11,16,18, 31, 33, 35, and 53) and one for a β-globin internal control marker. Two additional zip-codes were reserved for a pair of consensus HPV LDR products: the cLDR1 and cLDR2 primers hybridize to a conserved sequence within the HPV L1 open reading frame internal to the MY09/MY11 fragment. These consensus primers were shown to detect over 40 different HPV types. The purpose of this study was to evaluate the analytic performance of this low-density microarray based assay for HPV, as well as to introduce our simplified read-out instrumentation, shown here to be a low cost and highly efficient way to detect and genotype HPV for clinical testing.

Rapid, simultaneous genotyping of five common Southeast Asian beta-thalassemia mutations by multiplex minisequencing and denaturing HPLC.

β-Thalassemias are inherited hemoglobin disorders characterized by reduced production of β-globin chain. The severe forms of β-thalassemia produce marked anemia starting a few months after birth, and survival relies on regular blood transfusion and the lifelong use of drugs to prevent iron accumulation (1). β-Thalassemias are among the commonest genetic disorders in the world, and more than 200 mutations have been described to date. However, each at-risk population has a few common mutations (usually four or five) and a variable number of rare alleles (1). Common Southeast Asian β-thalassemia mutations include −28 A>G, codon (CD) 26 G>A, CD 41/42 −CTTT, CD 71/72 +A, IVS2 + 654 C>T, and others. The mutation CD 26 G>A produces hemoglobin E, presents like thalassemia, and is particularly common in Thailand, Cambodia, Vietnam, and Malaysia (1). There are many methods for genotyping known β-thalassemia mutations (2). Here we describe a rapid method for simultaneous genotyping of these five common mutations. The study was approved by the Human Subject Ethics Subcommittee of the Hong Kong Polytechnic University. The method is based on multiplex minisequencing followed by analysis of the single-base-extended products by denaturing HPLC (DHPLC). In minisequencing, a primer anneals to a DNA template immediately upstream of a mutation site to be analyzed and is extended by a single base as a result of incorporating a dideoxyribonucleotide triphosphate (ddNTP) (3). The single-stranded extension products are then separated by ion-pair reversed-phase liquid chromatography under completely denaturing condition, and the retention time is a function of both size and base composition (4). The DNA template for minisequencing was first generated by amplifying the whole human β-globin gene using primers HBBpF1 and HBBpR1 (Table 1⇓ ). The 25-μL reaction mixture contained 0.1 μM each of the primers, 50 ng of genomic DNA, 2.0 mM MgCl2 …

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands.

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus.

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.

Uniform 13C/15N-labeling of DNA by tandem repeat amplification

An optimized procedure has been described for the large-scale production of stable isotopeenriched duplex oligonucleotides of designed sequence. Large-scale production of labeled nucleotide triphosphates can be produced in this procedure simultaneously with labeled proteins, thereby providing synthetic dNMP precursors at no additional cost. The procedure is robust, with a minimum product:template yield of 800:1 overall, and produces > 99% single-length product. Tandem repeat PCR amplification is a general approach to large scale synthesis of duplex oligonucleotides and may have applications to both NMR and X-ray methods, particularly for product lengths in excess of 25 base pairs where failed sequences from solid-phase synthesis can be difficult to remove chromatographically. A drawback of the present approach is that the product is a duplex of two equal-length strands, making single-stranded products more difficult to prepare. For this reason, it could be preferable to produce single-stranded products by the [figure: see text] method of Zimmer and Crothers. Although a single base type can be selectively enriched in this approach, chemical synthesis will provide greater flexibility for labeled DNAs requiring site-specific labels at only one or a small number of nucleotide positions in the sequence. Therefore, maximum flexibility in labeling patterns can be realized by judicious choice of labeling method appropriate to the type of DNA product and extent of isotopic enrichment desired.

Direct DNA Sequencing of PCR Products

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam-Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by "asymmetric PCR," where one primer is present in vast excess over the other. This single-stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.

The MER3 Helicase Involved in Meiotic Crossing Over Is Stimulated by Single-stranded DNA-binding Proteins and Unwinds DNA in the 3′ to 5′ Direction

The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division. The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases. The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA. MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA. The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA. MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S. cerevisiae single-stranded DNA-binding protein, RPA (replication protein A). It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase. The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates. Possible roles for the MER3 helicase in meiotic crossing over are discussed.

Mapping psoralen cross-links at the nucleotide level in mammalian cells: suppression of cross-linking at transcription factor- or nucleosome-binding sites.

We have developed a new genomic sequencing method for detecting, with resolution at the nucleotide level, the interstrand DNA cross-links induced by 4,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cross-links (diadducts) initially formed are converted into monoadducts by alkali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR). After alkali reversal, but not before, the DNA strands can be separated and used as templates for gene-specific primer extension, which is the first step in the TD-PCR procedure. The converted psoralen adducts block primer extension, and the prematurely terminated single-stranded products are then amplified by TD-PCR and visualized on a sequencing gel. Adducts formed by angelicin, a psoralen derivative that forms only monoadducts, were also investigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vitro-treated DNA revealed that the binding of transcription factors inhibited both psoralen cross-linking and angelicin monoadduct formation in the c-JUN and c-FOS promoters in living human cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of general use for study of in vivo protein-DNA interactions and DNA repair.

Characterization of DNA damage at purine residues in oligonucleotides and calf thymus DNA induced by the mutagen 1-nitrosoindole-3-acetonitrile.

N-Nitrosoindoles can efficiently transfer the nitroso group to nucleophilic targets in isolated purine nucleotides, causing depurination, deamination, and the formation of a novel guanine analogue, oxanine [Lucas, L. T., Gatehouse, D., and Shuker, D. E. G. (1999) J. Biol. Chem. 274, 18319-18326]. To determine the likely biological relevance of these modification pathways, the reactivity of 1-nitrosoindole-3-acetonitrile (NIAN), a model 3-substituted N-nitrosoindole, with oligonucleotides and calf thymus DNA was examined at physiological pH and temperature. Reaction of NIAN with single-stranded oligonucleotides containing various guanine motifs resulted in the production of single-strand break products at guanine sites due to the formation of alkali-labile lesions. The number of lesions increased with NIAN concentration and incubation time. Modification of calf thymus DNA by NIAN resulted in depurination, which gave the corresponding purine bases, deamination coupled with depurination, which gave xanthine, and the formation of oxanine. The former pathway was clearly the most important, and all reaction products exhibited a dose-response relationship. Cytosine and thymine residues were inactive toward NIAN. Further studies revealed an additional product in NIAN-treated duplex DNA containing a CCGG motif that was characterized as an interstrand cross-link, the yield of which increased with increasing NIAN concentration. These results indicate that the transnitrosating ability of NIAN to modify purine residues is preserved at the macromolecular level, with guanine residues appearing to be a primary site of reaction. All of these modification processes are potentially mutagenic events if they occur in vivo.