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.     

Evidence for two mechanisms of palindrome-stimulated deletion in Escherichia coli: single-strand annealing and replication slipped mispairing

Spontaneous deletion mutations often occur at short direct repeats that flank inverted repeat sequences. Inverted repeats may initiate genetic rearrangements by formation of hairpin secondary structures that block DNA polymerases or are processed by structure-specific endonucleases. We have investigated the ability of inverted repeat sequences to stimulate deletion of flanking direct repeats in Escherichia coli. Propensity for cruciform extrusion in duplex DNA correlated with stimulation of flanking deletion, which was partially sbcD dependent. We propose two mechanisms for palindrome-stimulated deletion, SbcCD dependent and SbcCD independent. The SbcCD-dependent mechanism is initiated by SbcCD cleavage of cruciforms in duplex DNA followed by RecA-independent single-strand annealing at the flanking direct repeats, generating a deletion. Analysis of deletion endpoints is consistent with this model. We propose that the SbcCD-independent pathway involves replication slipped mispairing, evoked from stalling at hairpin structures formed on the single-stranded lagging-strand template. The skew of SbcCD-independent deletion endpoints with respect to the direction of replication supports this hypothesis. Surprisingly, even in the absence of palindromes, SbcD affected the location of deletion endpoints, suggesting that SbcCD-mediated strand processing may also accompany deletion unassociated with secondary structures.     

Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by S1 nuclease cleavage. Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule. Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property. Thus molecular size is unimportant for the S1 hypersensitivity of the Co1E1 inverted repeat. Furthermore, a 440 bp Sau3AI, EcoRI fragment of Co1E1 which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits S1 cleavage similar to Co1E1 in the supercoiled recombinant molecule. The effect is therefore both local and transmissible. Direct competition, between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated. Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising.     

Palindromy and the Location of Deletion Endpoints in Escherichia Coli

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.     

Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.     

Seven-Base-Pair Inverted Repeats in DNA Form Stable Hairpins in Vivo in Saccharomyces Cerevisiae

Palindromic sequences in single-stranded DNA and RNA have the potential for intrastrand base pairing, resulting in formation of "hairpin" structures. We previously reported a genetic method for detecting such structures in vivo in the yeast Saccharomyces cerevisiae. Below, we describe evidence indicating that a 14-base-pair palindrome (7 bp per inverted repeat) is sufficient for formation of a hairpin in vivo.     

The intragenomic polymorphism of a partially inverted repeat (PIR) in Gallus gallus domesticus, potential role of inverted repeats in satellite DNAs evolution

We report here the molecular characterization of the basic repeating unit of a novel repetitive family, partially inverted repeat (PIR), previously identified from chicken genome. This repetitive DNA family shares a close evolutionary relationship with XhoI/EcoRI repeats and chicken nuclear-membrane-associated (CNM) repeat. Sequence analyses reveal the 1430 bp basic repeating unit can be divided into two regions: the central region ( approximately 1000 bp) and the flanking region ( approximately 430 bp). Within the central region, a pair of repeats (86 bp) flanks the central core ( approximately 828 bp) in inversed orientation. Due to the tandem array feature shared by the repeating units, the inverted repeats fall between the central core and flanking region. Southern blot analyses further reveal the intragenomic polymorphism of PIR, and the molecular size of repeating units ranges from 1.1 kb to 1.6 kb. The identified monomer variants may result from multiple crossing-over events, implying the potential roles of inverted repeats in satellite DNAs variation.     

Long-range structural effects in supercoiled DNA: statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817-827] a set of sequences, called C-type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C-type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S-type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations: 1. the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C-type character; 2. C-type induction by an A + T rich Drosophila sequence; 3. low-temperature cruciform extrusion by an (AT)34 sequence; 4. the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat; 5. the effects of systematic deletion of the colL sequence; and 6. the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat. These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.     

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.     

A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

Nucleotide sequence of the maize transposable element Mul

A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.     

Deletion formation in bacteriophage T4

We have manipulated the dispensable region of the rIIB gene of bacteriophage T4 in order to study the generation of deletions involving direct repeats. We show that recombination between different parental chromosomes is one source of the deletions we have studied. We have also investigated the effects of structure, base composition and distance on deletion formation. We demonstrate that the potential to form structure in single-stranded DNA has variable effects on the frequency of deletion formation and conclude that, in some cases, slipped mispairing during DNA synthesis can make a substantial contribution to deletion frequencies. The G + C richness of the direct repeats involved in deletion formation is an important parameter of the frequency of deletion formation. We have confirmed that increasing the distance between direct repeats decreases deletion frequency.