Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site

Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence.     

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.     

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.     

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.     

A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles

Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.     

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.     

Rapid gene splicing and multi-sited mutagenesis by one-step overlap extension polymerase chain reaction

Gene splicing and site-directed mutagenesis (SDM) are important to introduce desired sequences in target DNA. However, introducing mutations at multiple sites requires multiple steps of DNA manipulation, which is time-consuming and labor-intensive. Here, we present a rapid efficient gene splicing and multi-sited mutagenesis method that introduces mutations at two distant sites via sequential connection of DNA fragments by one-step overlap extension polymerase chain reaction (OE-PCR). This bottom-up approach for DNA engineering can be broadly used to study protein structure-function, to optimize codon use for protein expression, and to assemble genes of interest.     

HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

To determine whether cellular replication factors can influence the fidelity of DNA replication, the effect of HeLa cell single-stranded DNA-binding protein (SSB) on the accuracy of DNA replication by HeLa cell DNA polymerase alpha has been examined. An in vitro gap-filling assay, in which the single-stranded gap contains the supF target gene, was used to measure mutagenesis. Addition of SSB to the in vitro DNA synthesis reaction increased the accuracy of DNA polymerase alpha by 2- to 8-fold. Analysis of the products of DNA synthesis indicated that SSB reduces pausing by the polymerase at specific sites in the single-stranded supF template. Sequence analysis of the types of errors resulting from synthesis in the absence or presence of SSB reveals that, while the errors are primarily base substitutions under both conditions, SSB reduces the number of errors found at 3 hotspots in the supF gene. Thus, a cellular replication factor (SSB) can influence the fidelity of a mammalian DNA polymerase in vitro, suggesting that the high accuracy of cellular DNA replication may be determined in part by the interaction between replication factors, DNA polymerase and the DNA template in the replication complex.     

Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round

Site-directed mutagenesis is a powerful tool to explore the structure-function relationship of proteins, but most traditional methods rely on the mutation of only one site at a time and efficiencies drop drastically when more than three sites are targeted simultaneously. Many applications in functional proteomics and genetic engineering, including codon optimization for heterologous expression, generation of cysteine-less proteins, and alanine-scanning mutagenesis, would greatly benefit from a multiple-site mutagenesis method with high efficiency. Here we describe the development of a simple and rapid method for site-directed mutagenesis of more than 10 sites simultaneously with up to 100% efficiency. The method uses two terminal tailed primers with a unique 25-nucleotide tail each that are simultaneously annealed to template DNA together with the set of mutagenic primers in between. Following synthesis of the mutant strand by primer extension and ligation with T4 DNA polymerase and ligase, the unique mutant strand-specific tails of the terminal primers are used as anchors to specifically amplify the mutant strand by high-fidelity polymerase chain reaction. We have employed this novel method to mutate simultaneously all 9 and 11 CUG leucine codons of the Hyg and Neo resistance genes, respectively, to the Candida albicans-friendly UUG leucine codon at 100% efficiency.     

Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli.

Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5'-pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli

The Klenow fragment structure, together with many biochemical experiments, has suggested a region of the protein that may contain the polymerase active site. We have changed 7 amino acid residues within this region by site-directed mutagenesis, yielding 12 mutant proteins which have been purified and analyzed in vitro. The results of steady-state kinetic determinations of Km(dNTP) and kcat for the polymerase reaction, together with measurements of DNA binding affinity, suggest strongly that this study has succeeded in targeting important active site residues. Moreover, the in vitro data allow dissection of the proposed active site region into two clusters of residues that are spatially, as well as functionally, fairly distinct. Mutations in Tyr766, Arg841, and Asn845 cause an increase in Km(dNTP), suggesting that contacts with the incoming dNTP are made in this region. Mutations in the second cluster of residues, Gln849, Arg668, and Asp882, cause a large decrease in kcat, suggesting a role for these residues in catalysis of the polymerase reaction. The DNA-binding properties of mutations at positions 849 and 668 may indicate that the catalytic role of these side chains is associated with their interaction with the DNA substrate. Screening of the mutations in vivo for the classical polA-defective phenotype (sensitivity to DNA damage) demonstrated that a genetic screen of this type may be a reasonable predictor or kcat or of DNA binding affinity in future mutational studies.     

Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

A modified polymerase chain reaction (PCR)-based site-directed mutagenesis method used to splice together different regions of a gene by deleting hundreds of nucleotides of undesired sequences is described. This method was inspired by a PCR-based site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA); the procedure and primer design were modified to enable the method to generate deletions several hundreds of nucleotides in length with an efficiency of 80-100%, and to delete two DNA fragments simultaneously in a single PCR. This method should be useful for deletion of large DNA fragments from a gene.     

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.     

The relative roles in vivo of Saccharomyces cerevisiae Pol eta, Pol zeta, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer

We have investigated the relative roles in vivo of Saccharomyces cerevisiae DNA polymerase eta, DNA polymerase zeta, Rev1 protein, and the DNA polymerase delta subunit, Pol32, in the bypass of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer, by transforming strains deleted for RAD30, REV3, REV1, or POL32 with duplex plasmids carrying one of these DNA lesions located within a 28-nucleotide single-stranded region. DNA polymerase eta was found to be involved only rarely in the bypass of the T-T (6-4) photoadduct or the abasic sites in the sequence context used, although, as expected, it was solely responsible for the bypass of the T-T dimer. We argue that DNA polymerase zeta, rather than DNA polymerase delta as previously suggested, is responsible for insertion in bypass events other than those in which polymerase eta performs this function. However, DNA polymerase delta is involved indirectly in mutagenesis, since the strain lacking its Pol32 subunit, known to be deficient in mutagenesis, shows as little bypass of the T-T (6-4) photoadduct or the abasic sites as those deficient in Pol zeta or Rev1. In contrast, bypass of the T-T dimer in the pol32delta strain occurs at the wild-type frequency.     

The role of DNA polymerase iota in UV mutational spectra

UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol iota). In order to clarify the specific role of Pol iota in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol eta. Synthetic RNA duplexes were used to efficiently inhibit Pol iota expression in 293 T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293 T cells in presence of anti-Pol iota siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol iota knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol iota does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol iota has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.     

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.     

Holoenzyme DNA polymerase III fixes mutations

DNA polymerase III is required for mutagenesis after damage to the chromosome. This effect is not modulated by the presence or absence of DNA polymerase II activity in the cell. In cells containing a temperature-sensitive dnaE mutation, the alpha-subunit of DNA polymerase III is inactivated at the restrictive temperature, resulting in lethality. Cells containing the pcbA1 mutation can continue replication if DNA polymerase I activity is present. When such cells are shifted from the permissive to the restrictive temperature, mutagenesis decreases rapidly after 10 min. These results are compatible with conversion of the replicative apparatus from one containing a functional DNA polymerase III synthetic subunit to one containing DNA polymerase I. We also find that DNA polymerase I dependent replication is markedly sensitive to coumermycin A1. We conclude that DNA polymerase III holoenzyme with the alpha-subunit is required for fixing mutations in the genome.     

The Specificity of Topoisomerase-Mediated DNA Cleavage Defines Acridine-Induced Frameshift Specificity within a Hotspot in Bacteriophage T4

Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.     

Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.

The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.