Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a −1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.     

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.     

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.     

A Highly Revertible Cyc1 Mutant of Yeast Contains a Small Tandem Duplication

A mutant, cyc1-96, that reverts spontaneously at an extremely high rate, was uncovered after examining approximately 500 cyc1 mutants which lack or have defective iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Cloning and DNA sequencing of appropriate fragments revealed that the cyc1-96 mutation contained a 19 bp duplication whereas the spontaneously arising revertants contained the normal wild-type sequence. Because the 19 bp segment in the wild-type sequence is flanked by a 5 bp repeat and because the cyc1-96 mutation arose spontaneously, the 19 bp duplication may have arisen by slippage and misalignment during DNA synthesis. The high reversion rate was not diminished in strains containing the rad52 mutation, which generally reduces mitotic recombination, including recombination associated with the elimination of a segment of a long direct repeat. Thus the loss of segments from short and long duplications occur by different mechanisms. We suggest that the high reversion rates of cyc1-96 and other short duplications are due to misalignment errors during replication.     

Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae.

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A.T----G.C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G.C----A.T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G.C----A.T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A.T----G.C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.     

Mutator Phenotype Induced by Aberrant Replication

We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase δ (polδ) and mutants of Polα, two Polδ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2⁺, which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2Δ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2Δ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2Δ. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.     

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.     

Differential DNA secondary structure-mediated deletion mutation in the leading and lagging strands.

The frequencies of deletion of short sequences (mutation inserts) inserted into the chloramphenicol acetyl-transferase (CAT) gene were measured for pBR325 and pBR523, in which the orientation of the CAT gene was reversed, in Escherichia coli. Reversal of the CAT gene changes the relationship between the transcribed strand and the leading and lagging strands of the DNA replication fork in pBR325-based plasmids. Deletion of these mutation inserts may be mediated by slipped misalignment during DNA replication. Symmetrical sequences, in which the same potential DNA structural misalignment can form in both the leading and lagging strands, exhibited an approximately twofold difference in the deletion frequencies upon reversal of the CAT gene. Sequences that contained an inverted repeat that was asymmetric with respect to flanking direct repeats were designed. With asymmetric mutation inserts, different misaligned structural intermediates could form in the leading and lagging strands, depending on the orientation of the insert and/or of the CAT gene. When slippage could be stabilized by a hairpin in the lagging strand, thereby forming a three-way junction, deletion occurred by up to 50-fold more frequently than when this structure formed in the leading strand. These results support the model that slipped misalignment involving DNA secondary structure occurs preferentially in the lagging strand during DNA replication.     

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

Regional genomic instability predisposes to complex dystrophin gene rearrangements

Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.     

Mutagenesis assays in yeast

Analyzing mutation spectra is a very powerful method to determine the effects of various types of DNA damage and to understand the workings of various DNA repair pathways. However, compiling sequence-specific mutation spectra is laborious; even with modern sequencing technology, it is rare to obtain spectra with more than several hundred data points. Two assay systems are described for yeast, one for insertion/deletion mutations and one for base substitution mutations, that allow determination of specific mutations without the necessity of DNA sequencing. The assay for insertion/deletion mutations uses a variety of different simple repeats placed in frame with URA3 such that insertions or deletions lead to a selectable Ura(-) phenotype; essentially all such mutations are in the simple repeat sequence. The assay for base substitution mutations uses a series of six strains with different mutations in one essential codon of the CYC1 gene. Because only true reversions lead to a selectable phenotype, the bases mutated in any reversion event are known. The advantage of these assays is that they can quantitatively determine over several orders of magnitude the types of mutations that occur under a given set of conditions, without DNA sequencing.     

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.     

Pure exogenous singlet oxygen: nonmutagenicity in bacteria

Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.     

Replication restart: a pathway for (CTG).(CAG) repeat deletion in Escherichia coli

(CTG)n.(CAG)n repeats undergo deletion at a high rate in plasmids in Escherichia coli in a process that involves RecA and RecB. In addition, DNA replication fork progression can be blocked during synthesis of (CTG)n.(CAG)n repeats. Replication forks stalled at (CTG)n.(CAG)n repeats may be rescued by replication restart that involves recombination as well as enzymes involved in replication and DNA repair, and this process may be responsible for the high rate of repeat deletion in E. coli. To test this hypothesis (CAG)n.(CTG)n deletion rates were measured in several E. coli strains carrying mutations involved in replication restart. (CAG)n.(CTG)n deletion rates were decreased, relative to the rates in wild type cells, in strains containing mutations in priA, recG, ruvAB, and recO. Mutations in priB and priC resulted in small reductions in deletion rates. In a recF strain, rates were decreased when (CAG)n comprised the leading template strand, but rates were increased when (CTG)n comprised the leading template. Deletion rates were increased slightly in a recJ strain. The mutational spectra for most mutant strains were altered relative to those in parental strains. In addition, purified PriA and RecG proteins showed unexpected binding to single-stranded, duplex, and forked DNAs containing (CAG)n and/or (CTG)n loop-outs in various positions. The results presented are consistent with an interpretation that the high rates of trinucleotide repeat instability observed in E. coli result from the attempted restart of replication forks stalled at (CAG)n.(CTG)n repeats.     

On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.     

Molecular analysis of the complete set of length mutations found in the plastomes of Triticum-Aegilops species.

Precise location and nature of each of 14 length mutations detected among chloroplast DNAs of Triticum-Aegilops species by RFLP analysis were determined at the nucleotide sequence level. Each mutation was compared with at least three non-mutated wild-type plastomes as standards. These 14 length mutations were classified into 4 duplications and 10 deletions. One duplication occurred in the small single-copy region close to the border of the inverted repeat, and the remaining 13 length mutations took place in the large single-copy region. All length mutations occurred in the intergenic regions, suggesting that these length mutations do not affect plastid gene expression. Saltatory replication was the cause of all duplications, whereas intramolecular recombination mediated by short direct repeats played a substantial role in the deletions. Recurrent occurrences of certain deletion events were found in some AT-rich regions, which constituted hot spots for deletion. Out of four hypervariable regions detected among the grass plastomes, two (downstream of rbcL and a tRNA gene accumulated region) were still active after differentiation of Triticum and Aegilops complex.