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.     

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.     

A simple vector modification to facilitate oligonucleotide-directed mutagenesis.

We describe a simple modification of commonly used single-stranded cloning vectors that permits the efficient recovery of mutant DNA molecules in oligonucleotide-directed mutagenesis experiments, even when the absolute efficiency of mutagenesis is very low. The modification consists of the insertion of a short synthetic DNA fragment into the vector's polylinker and permits the identification of mutant clones based on a standard chromogenic plate assay for bacterial colonies or phage plaques producing functional beta-galactosidase. Other useful properties of the original vector are retained in the modified version. In vitro mutagenesis reactions are carried out with two oligonucleotides, one to introduce the mutation of interest, and the second to correct a frameshift mutation introduced into the beta-galactosidase gene of the modified vector. We have found that these two sequence changes are closely linked following transformation of an appropriate E. coli strain with the products of the in vitro mutagenesis reaction, and have thereby recovered desired mutations at a frequency of about 50% even when the overall mutagenesis efficiency is less than 1%. By alternately correcting and re-introducing the beta-galactosidase frameshift mutation, we have shown that multiple rounds of mutagenesis can be carried out on the same template with a high efficiency of mutant recovery in each step. Modifications similar or identical to those we describe here should be feasible for most commonly used single-stranded cloning vectors and should increase the usefulness of these vectors by providing an additional option for oligonucleotide-directed mutagenesis to be used in conjunction with or in lieu of other commonly used approaches.     

Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions.

Human DNA polymerase eta (hPol eta) is one of the newly identified Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPol eta accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the fingers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identifies Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPol eta S62G, a mutant polymerase with enhanced activity, to bypass five other site-specific lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.     

Mutagenic spectrum resulting from DNA damage by oxygen radicals.

Oxygen free radicals are highly reactive species that damage DNA and cause mutations. We determined the mutagenic spectrum of oxygen free radicals produced by the aerobic incubation of single-stranded M13mp2 DNA with Fe2+. The Fe2(+)-treated DNA was transfected into component Escherichia coli, and mutants within the nonessential lac Z alpha gene for beta-galactosidase were identified by decreased alpha-complementation. The frequency of mutants obtained with 10 microM Fe2+ was 20- to 80-fold greater than that obtained with untreated DNA. Mutagenesis was greater after the host cells were exposed to UV irradiation to induce the SOS "error-prone" response. The ability of catalase, mannitol, and superoxide dismutase to diminish mutagenesis indicates the involvement of oxygen free radicals. The sequence data on 94 of the mutants establish that mutagenesis results primarily from an increase in single-base substitutions. Ninety-four percent of the mutants with detectable changes in nucleotide sequence were single-base substitutions, the most frequent being G----C transversions, followed by C----T transitions and G----T transversions. The clustering of mutations at distinct gene positions suggests that Fe2+/oxygen damage to DNA is nonrandom. This mutational spectrum provides evidence that a multiplicity of DNA lesions produced by oxygen free radicals in vitro are promutagenic and could be a source of spontaneous mutations.     

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5,6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at AT or GC pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide–DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.     

Reconstruction of DNA sequences using genetic algorithms and cellular automata: Towards mutation prediction?

Change of DNA sequence that fuels evolution is, to a certain extent, a deterministic process because mutagenesis does not occur in an absolutely random manner. So far, it has not been possible to decipher the rules that govern DNA sequence evolution due to the extreme complexity of the entire process. In our attempt to approach this issue we focus solely on the mechanisms of mutagenesis and deliberately disregard the role of natural selection. Hence, in this analysis, evolution refers to the accumulation of genetic alterations that originate from mutations and are transmitted through generations without being subjected to natural selection. We have developed a software tool that allows modelling of a DNA sequence as a one-dimensional cellular automaton (CA) with four states per cell which correspond to the four DNA bases, i.e. A, C, T and G. The four states are represented by numbers of the quaternary number system. Moreover, we have developed genetic algorithms (GAs) in order to determine the rules of CA evolution that simulate the DNA evolution process. Linear evolution rules were considered and square matrices were used to represent them. If DNA sequences of different evolution steps are available, our approach allows the determination of the underlying evolution rule(s). Conversely, once the evolution rules are deciphered, our tool may reconstruct the DNA sequence in any previous evolution step for which the exact sequence information was unknown. The developed tool may be used to test various parameters that could influence evolution. We describe a paradigm relying on the assumption that mutagenesis is governed by a near-neighbour-dependent mechanism. Based on the satisfactory performance of our system in the deliberately simplified example, we propose that our approach could offer a starting point for future attempts to understand the mechanisms that govern evolution. The developed software is open-source and has a user-friendly graphical input interface.     

Multiple Site Directed Mutagenesis Strategy Based on Total RNA and RT-PCR Method

Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility of this strategy by mutation of the human β-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed mutagenesis.     

Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.     

"DIROM"--an interactive system for planning experiments on directed mutagenesis and design of artificial genes]

We present a computer system "DIROM" for oligonucleotide-directed mutagenesis and artificial gene design experiments planning and support. "DIROM" allows to search for optimal oligonucleotides according to such parameters as sufficient energy of oligonucleotide-target hybridization, secondary structure of oligonucleotide and target DNA, presence of alternative attachment sites in target DNA, terminal G/C pairs presence. Both single-stranded and double-stranded vector mutagenesis methods are implemented. It can be also used for optimal primer selection for polymerase chain reaction, sequencing etc. "DIROM" can search for both existent and potential carry out vector+target sequence construction. Both amino acid and nucleotide sequences can be operated.     

DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to generate every possible mutation (36 in all) within 11 base-pairs of one FLP protein binding site and the base-pair immediately flanking it. The base-pairs within the binding site can be separated into three classes on the basis of these results. Thirty of the 36 sequence changes, including all three at seven different positions (class I) produce a negligible or modest effect on FLP protein-promoted recombination. In particular, most transition mutations are well-tolerated in this system. In only one case do all three possible mutations produce large effects (class II). At three positions, clustered near the site at which DNA is cleaved by FLP protein, one of the two possible transversions produces a large effect on recombination, while the other two changes produce modest effects (class III). For seven mutants for which FLP protein binding was measured, a direct correlation between decreases in recombination activity and in binding was observed. Positive effects on the reaction potential of mutant sites are observed when the other FLP binding site in a single recombination site is unaltered or when the second recombination site in a reaction is wild-type. This suggests a functional interaction between FLP binding sites both in cis and in trans. When two mutant recombination sites (each with 1 altered FLP binding site) are recombined, the relative orientation of the mutations (parallel or antiparallel) has no effect on the result. These results provide an extensive substrate catalog to complement future studies in this system.     

Lack of strand bias in UV-induced mutagenesis in Escherichia coli

We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.     

DNA binding is required for the apoptogenic action of apoptosis inducing factor

The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.     

Spectrum of N4-aminocytidine mutagenesis

N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.     

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.     

Mutation spectrum of copper-induced DNA damage.

The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.     

Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.

We describe a modification of the polymerase chain reaction technique which allows amplification of cellular DNA adjacent to an integrated provirus given sequence information for the provirus only. The modified technique should be generally useful for studies of insertional mutagenesis and other situations in which one wishes to isolate DNA adjacent to a region of known sequence.     

Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis

We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis. Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-β. In the presence of either polymerase- or E. coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen. Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations. We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-β).     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.     

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)