A novel mutational hotspot in a natural quasipalindrome in Escherichia coli

We have found that most spontaneous mutations in the thyA gene of Escherichia coli selected for resistance to trimethoprim result from a TA to AT transversion at a single site within an imperfect inverted repeat or quasipalindrome sequence. This natural quasipalindrome within the coding region of thyA contains an extraordinarily potent hotspot for mutation. Our analysis provides evidence that these mutations are templated by nearby sequences by replication within a hairpin structure. Although quasipalindrome-associated mutations have been observed in many organisms, including humans, the cellular avoidance mechanisms for these unusual mutational events have remained unexplored. We find that the mutational hotspot in thyA is dramatically stimulated by inactivation of exonucleases I and VII, which degrade single-strand DNA with a common 3'-5' polarity. We propose that these exonucleases abort the replicative misalignment events that initiate hairpin-templated mutagenesis by degrading displaced nascent DNA strands. Mismatch repair-defective strains also showed increased mutability at the hotspot, consistent with the notion that these mutations arise during chromosomal lagging-strand replication and are often subsequently removed by methyl-directed mismatch repair. The absence of the thyA quasipalindrome sequence from other related bacterial genera suggests that this sequence represents a "selfish" DNA element whose existence itself is driven by this unusual hairpin-templating mechanism.     

Theoretical analysis of mechanisms of spontaneous and induced mutations in DNA based on repeated sequences

Mechanisms of spontaneous and chemically induced point mutations' emergence in DNA have theoretically been investigated using the statistical weight method. We have analysed 12 nucleotide sequences containing 95 point spontaneous mutations and 3 sequences comprising 30 mutations induced by such mutagens as 4'-hydroxymethyl-4,5',8-threemethylpsoralene, natrium bisulfite, hydroxylamine. The possibility of occurrence of point mutations by repair correction of heteroduplexes formed via mispairing of imperfect direct and inverted repeats in DNA has been studied. Statistically reliable connection of position of spontaneous mutations in DNA with repeats has been revealed for a number of nucleotide sequences. Statistically reliable connection of mutations induced by 4'-hydroxymethyl-4,5',8-threemethylpsoralene with imperfect repeats is also shown.     

Mutations predetermined by the primary structure of DNA

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.     

Cloning of the vaccinia virus telomere in a yeast plasmid vector

The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector. Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome. As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication. Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini.     

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.     

Molecular cloning of the terminal hairpin of vaccinia virus DNA as an imperfect palindrome in an Escherichia coli plasmid

The genome of vaccinia virus is a linear duplex molecule of approximately 185 kb with hairpins at each end that link the complementary strands. The hairpins, which exist in two forms that are inverted and complementary in sequence, were isolated as XbaI restriction fragments and converted to a linear intermolecular duplex structure by denaturation and reannealing. The latter was then stably cloned as a 142-bp imperfect palindrome in an Escherichia coli plasmid. The insert was excised from the plasmid and the palindrome was extended on both sides by ligating it to the adjacent vaccinia virus DNA segment. The resulting fragment was cloned as a 278-bp imperfect palindrome. Restriction endonuclease analysis and DNA sequencing indicated the absence of any deletions or rearrangements. After excision from the plasmid, the palindrome was converted by heating and rapid cooling to the original two hairpin forms. In this manner, large quantities of vaccinia virus telomeres may be obtained for physical and biochemical studies.     

Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions

Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.     

Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.     

Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini.

The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres.     

Cloning yeast telomeres on linear plasmid vectors

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast. The fact that yeast can recognize and use DNA ends from the distantly related organism Tetrahymena suggests that the structural features required for telomere replication and resolution have been highly conserved in evolution. The linear plasmid was used as a vector to clone chromosomal telomeres from yeast. One Tetrahymena end was removed by restriction digestion, and yeast fragments that could function as an end on a linear plasmid were selected. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres, and suggested that all yeast chromosomes might have a common telomere sequence. Yeast telomeres appear to be similar in structure to the rDNA of Tetrahymena, in which specific nicks or gaps are present within a simple repeated sequence near the terminus of the DNA.     

Gene Correction by RNA‐DNA Oligonucleotides

An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate the correction of single‐base mutations of episomal and chromosomal targets in mammalian cells. The design of the oligonucleotide exploited the highly recombinogenic RNA‐DNA hybrids and featured hairpin capped ends avoiding destruction by cellular helicases or exonucleases. The RNA‐DNA oligonucleotide (RDO) was designed to correct a point mutation in the tyrosinase gene and caused a permanent gene correction in mouse albino melanocytes, determined by clonal analysis at the level of genomic sequence, protein and phenotypic change. Recently, we demonstrated correction of the tyrosinase gene using the same RDO in vivo, as detected by dark pigmentation of several hairs and DOPA staining of hair follicles in the treated skin of albino mice. Such RDOs might hold a promise as a therapeutic method for the treatment of skin diseases. However, the frequency of gene correction varies among different cells, indicating that cellular activities, such as recombination and repair, may be important for gene conversion by RDOs. As this technology becomes more widely utilized in the scientific community, it will be important to understand the mechanism and to optimize the design of RDOs to improve their efficiency and general applicability.     

In Vitro Direct Repeats-mediated Deletion During PCR Amplification

While conducting our research on mutations in the human blood platelet glycoprotein Ib-alpha (GPIbalpha) gene, we detected an unusual deletion of 84 bp. This deletion took place in vitro, during PCR and between two direct repeats. It was observed that the deletion could be detected either by the direct sequencing of the PCR product or after the latter's cloning into a plasmid. After observing a series of four sequenced clones from the same individual, we noticed that while three had the same 84-bp deletion, the fourth exhibited a shorter one. We also noted that there were no cases wherein both deleted and undeleted amplicons coexisted and that several point mutations occurred in the sequence surrounding the deletion. Such Taq errors are statistically more frequent in the "deletion prone DNA" than usual. Interestingly, the deletion was observed only in a DNA, which we call here "deletion prone DNA", whose structure might have been particularly reorganized. Indeed, the mung bean nuclease pre-treatment of this DNA prior to PCR prevented the deletion, thus strengthening the hypothesis that an intra-strand hairpin structure was involved in the deletion process. Direct repeats-mediated deletion is well known in vivo but this is the first report of such "in vitro direct repeats deletion".     

The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks.

The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT .     

Induction of repetitive nucleotide sequences. The probable mechanisms of genome evolution and gene conversion

In the preselected site of pBR322 plasmid DNA related to the Tcr gene mutations were induced by the complementary single-stranded DNA restricts carrying alkylating groups. The alterations of the DNA primary structure in the mutagenized site were studied. It was found and that in the majority of mutants with the impaired Tcr gene function, the tandem direct repeats appeared. The repeats of 7-8 base pairs were localized in a fixed site of the Tcr gene, downstream of the palindrome. It is suggested that tandem repeats appear as a result of D-loops formation when single-stranded DNA forms a hairpin structure, due to the presence of palindromes. In the light of this notion, the tentative schemes of gene conversion and genome evolution are discussed.     

Precise determination, cross-recognition, and functional analysis of the double-strand origins of the rolling-circle replication plasmids in haloarchaea

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO_Z) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.     

Nucleotide sequence required for resolution of the concatemer junction of vaccinia virus DNA.

The mature form of the vaccinia virus genome consists of a linear, 185,000-base-pair (bp) DNA molecule with a 10,000-bp inverted terminal repetition and incompletely base-paired 104-nucleotide hairpin loops connecting the two strands at each end. In concatemeric forms of intracellular vaccinia virus DNA, the inverted terminal repetitions of adjacent genomes form an imperfect palindrome. The apex of this palindrome corresponds in sequence to the double-stranded form of the hairpin loop. Circular plasmids containing palindromic concatemer junction fragments of 250 bp or longer are converted into linear minichromosomes with hairpin ends when they are transfected into vaccinia virus-infected cells, providing a model system with which to study the resolution process. To distinguish between sequence-specific and structural requirements for resolution, plasmids with symmetrical insertions, deletions, and oligonucleotide-directed mutations within the concatemer junction were constructed. A sequence (ATTTAGTGTCTAGAAAAAAA) located on both sides of the apex segment was found to be critical for resolution. Resolution was more efficient when additional nucleotides, TGTG, followed the run of A residues. Both the location and sequence of the proposed resolution signal are highly conserved among poxviruses.     

Double displacement loops (double d-loops) are templates for oligonucleotide-directed mutagenesis and gene repair

Appreciable levels of gene repair result from the hybridization of two oligonucleotides at a specific site in a mutated gene and subsequent correction by a form of oligonucleotide-directed mutagenesis known as gene repair. The incorporation of the two oligonucleotides into superhelical plasmid DNA leads to the formation of double d-loops, structures shown to be templates for the repair of both frameshift and point mutations. Structural limitations placed on the template indicate that correction is influenced significantly by the positioning of the second oligonucleotide, known as the annealing oligonucleotide. Complexes constructed with two oligonucleotides directly opposite each other exhibit the highest levels of gene repair activity. Blocking the 3'-end of either oligonucleotide with an amino C7 group does not diminish the performance of the double d-loop as a template for correction of the point mutation, suggesting that primer extension does not play a pivotal role in the mechanism of gene repair.     

用改进的重叠延伸PCR技术构建三位点突变载体

目的:改进传统重叠延伸PCR方法,实现引入3个不同DNA突变位点的简便的多位点定点突变。方法:根据前期构建的包含人线粒体12S rRNA(NC 01290)3个热点突变位点的野生型质粒序列,利用Muta Primer 2.0软件设计针对3个热点突变位点的3对互补的定点突变引物,以野生型质粒为模板,结合重叠延伸PCR反应和冷冻析出法,产生同时包含3个突变位点的突变目的片段,酶切后克隆到载体中,测序确证是否突变成功。结果:DNA测序证实3个不同突变位点同时成功引入,定点突变载体构建成功。结论:用改进的重叠延伸PCR技术能简便、高效地获得多位点定点突变载体,在分子生物学领域有较高的使用价值。     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.