A one-step method for quantitative determination of uracil in DNA by real-time PCR

Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome.     

Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

A rapid site-directed mutagenesis strategy using homologous recombination and DpnI digestion of the template in Escherichia coli is described. Briefly, inverse polymerase chain reaction amplification of the entire circular plasmid was performed by mutagenic primers with overlapping sequences ( approximately 15 bp) for generating PCR products with approximately 15 bp of homology on the terminal ends. On direct transformation of the amplified PCR products into restriction endonuclease DpnI-expressing E. coli BUNDpnI, homologous recombination occurs in E. coli while the original templates are removed via DpnI digestion in vivo, thus yielding clones harboring mutated circular plasmids. Nearly 100% efficiency was attained when this strategy was used to modify DNA sequences.     

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.     

Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI

DNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N(6)-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick.     

Transfer of recombinant plasmids containing the gene for DpnII DNA methylase into strains of Streptococcus pneumoniae that produce DpnI or DpnII restriction endonucleases.

Plasmid transfer via the transformation pathway of Streptococcus pneumoniae was weakly restricted by the DpnI or DpnII restriction endonuclease, either of which gave a reduction only to 0.4, compared with phage infection, which was restricted to 10(-5). The greater sensitivity of plasmid transfer compared with chromosomal transformation, which was not at all restricted, can be attributed to partially double-stranded intermediates formed from two complementary donor fragments. However, clustering of potential restriction sites in the plasmids increased the probability of escape from restriction. The recombinant plasmid pMP10 , in which the gene for the DpnII DNA methylase was cloned, can be transferred to strains that contain neither restriction enzyme or that contain DpnII as readily as can the vector pMP5 . Introduction of pMP10 raised the level of methylase by five times the level normally present in DpnII strains. Transfer of pMP10 to DpnI -containing strains was infrequent, presumably owing to the suicidal methylation of DNA which rendered it susceptible to the host endonuclease. The few clones in which pMP10 was established had lost DpnI . Loss of the plasmid after curing of the cell eliminated the methylase but did not restore DpnI . Although this loss of DpnI could result from spontaneous mutation, its relatively high frequency, 0.1% suggested that the loss was due to a regulatory shift.     

Transformation of restriction endonuclease phenotype in Streptococcus pneumoniae

The genetic basis of the unique restriction endonuclease DpnI, that cleaves only at a methylated sequence, 5'-GmeATC-3', and of the complementary endonuclease DpnII, which cleaves at the same sequence when it is not methylated, was investigated. Different strains of Streptococcus pneumoniae isolated from patients contained either DpnI (two isolates) or DpnII (six isolates). The latter strains also contained DNA methylated at the 5'-GATC-3' sequence. A restrictable bacteriophage, HB-3, was used to characterize the various strains and to select for transformants. One laboratory strain contained neither DpnI nor Dpn II. It was probably derived from a DpnI-containing strain, and its DNA was not methylated at 5'-GATC-3'. Cells of this strain were transformed to the DpnI restriction phenotype by DNA from a DpnI-containing strain and to the DpnII restriction phenotype by DNA from a DpnII-containing strain. Neither cross-transformation, that is, transformation to one phenotype by DNA from a strain of the other phenotype, nor spontaneous conversion was observed. Extracts of transformants to the new restriction phenotype were shown to contain the corresponding endonuclease.     

Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism

Cells of S. pneumoniae contain either DpnI, a restriction endonuclease that cleaves only the methylated DNA sequence 5'-GmeATC-3', or DpnII, which cleaves the same sequence when not methylated. A chromosomal DNA segment containing DpnII genes was cloned in S. pneumoniae. Nucleotide sequencing of this segment revealed genes encoding the methylase and endonuclease and a third protein of unknown function. When the plasmid was introduced into DpnI cells, recombination during chromosomal facilitation of its establishment substituted genes encoding the DpnI endonuclease and another protein in place of the DpnII genes. DNA hybridization and sequencing showed that the DpnI and DpnII segments share homology on either side but not between themselves or with other regions of the chromosome. Thus, the complementary restriction systems are found on nonhomologous and mutually exclusive cassettes that can be inserted into a particular point in the chromosome of S. pneumoniae on the basis of neighboring homology.     

Structural basis of the methylation specificity of R.DpnI

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.     

In vitro mutagenesis as a result of 60Co-gamma-ray-induced base damage.

We utilized a model system to study the mechanism(s) of mutation resulting from gamma-ray-induced DNA base damage. 60Co-irradiated, uracil-containing M13mp2 DNA was hybridized to normal (non-uracil) linearized double-stranded virus DNA minus the lac reporter region. Only DNA without strand breaks in the reporter region will circularize. This DNA was used as a substrate for a modified T7 DNA polymerase with no residual 3'----5' exonuclease activity (Sequenase 2). The reaction product was transfected into a rec- bacterial host to minimize the occurrence of bypass events in vivo, and mutant progeny were selected. DNA irradiated with 400 or 800 Gy from a 60Co gamma-source gave about a 5-fold increase in the percentage of mutants recovered after synthesis with Sequenase as compared to the recovery of mutants using control DNA. About 20% of the mutants recovered from both irradiated and control templates contained multiple mutations in the target area sequenced. The irradiated samples had an excess of mutations which resulted from changes at pyrimidines. C---T transitions were most common. Mutations at T were mostly (-1) and (-2) frameshifts, particularly at sequences of repeated T's.     

Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease

Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.     

Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies

PCR analyses of ancient and degraded DNA suffer from their extreme sensitivity to contamination by modern DNA originating, in particular, from carryover contamination with previously amplified or cloned material. Any strategy for limiting carryover contamination would also have to be compatible with the particular requirements of ancient DNA analyses. These include the need (i) to amplify short PCR products due to template fragmentation; (ii) to clone PCR products in order to track possible base misincorporation resulting from damaged templates; and (iii) to avoid incomplete decontamination causing artifactual sequence transformation. Here we show that the enzymatic decontamination procedures based upon dUTP- and uracil-N-glycosylase (UNG) can be adapted to meet the specific requirements of ancient DNA research. Thus, efficiency can be improved to vastly reduce the amplification of fragments < or = 100 bp. Secondly, the use of an Escherichia coli strain deficient in both UNG and dUTPase allows for the cloning of uracil-containing PCR products and offers protection from plasmid DNA contamination, and, lastly, PCR products amplified from UNG-degraded material are free of misleading sequence modifications.     

Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI G^m6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.     

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae.

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.     

Bacteriophage PBS2-induced inhibition of uracil-containing DNA degradation.

Degradation of uracil-containing DNA by Bacillus subtilis extracts and its inhibition after infection by the uracil-containing DNA phage PBS2 have been investigated to resolve differences between the published reports of Tomita and Takahashi (1975) and Friedberg et al. (1975, 1976). The product of hydrolysis of PBS2 DNA, tritium labeled in its uracil and cytosine residues, is solely uracil and not deoxyuridine. The degrading activity is completely inhibited within 7 min after PBS2 infection, before any other known PBS2-induced protein is detectable. The production of the PBS2 inhibitor (a small, heat-stable protein) continues until 10 to 20 min postinfection.     

Cleavage at the twelve-base-pair sequence 5''-TCTAGATCTAGA-3'' using M.Xbal (TCTAGm6A) methylation and DpnI (Gm6A/TC) cleavage.

The DNA methylase M.Xbal was isolated from an E. coli recombinant clone. We deduce that the enzyme methylates at the sequence 5'-TCTAGm6A-3'. In combination with the methylation-dependent restriction endonuclease, DpnI (5'-Gm6A/TC-3'), DNA cleavage occurs at the sequence 5'-TCTAGA/TCTAGA-3'. This twelve-base-pair site should occur once every 16,000,000 base pairs in a random sequence of DNA. The exceptional rarity of the M.XbaI/DpnI sequence makes it an ideal candidate for transpositional integration of a unique cleavage site into bacterial genomes. Retrotransposition into mammalian genomes is also an attractive possibility.     

Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.     

Random DNA fragmentation with endonuclease V: application to DNA shuffling

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3′ to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.     

Evidence that Escherichia coli virus T1 induces a DNA methyltransferase

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.     

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.     

Recombination of uracil-containing lambda bacteriophages.

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.