Singular over-representation of an octameric palindrome, HIP1, in DNA from many cyanobacteria.

An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome). The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries. DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1. Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage. To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced. HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides. An oligonucleotide including HIP1 was also tested in PCR. Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns. However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.     

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid Pbr322

The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.     

Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae

Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.     

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.     

Effects of palindrome size and sequence on genetic stability in the bacteriophage phi X174 genome

By inserting palindromes of varying length and sequence into a non-essential region of the bacteriophage phi X174 genome we have investigated the effect of palindrome size and sequence on their genetic stability. Multimers of increasing size of the EcoRI linker CCGAATTCGG (E), the BamHI linker CCGGATCCGG (B) or mixtures of both (E, B) were inserted into the PvuII site of a previously constructed bacteriophage strain phi X174 J-F ins6. The largest inserts that could be maintained in the genome without significant loss of genetic stability were 2B, 4E, and 4(E, B), respectively. Polymers exceeding this size could be inserted but resulted in rapid and precise deletion from the phage genome, whereby nB was more unstable than nE, and nE was more unstable than n(E, B). Analysis of the resulting deletion mutants provided evidence for two different types of deletions. The more frequent deletion arose from either type palindrome and removed nucleotides in blocks of ten base-pairs (one linker unit), but only from the palindromic sequence, and always left at least an 18 base-pair long palindrome (one linker plus 8 neighboring base-pairs) behind. The less frequently occurring deletions arose only from nB type palindromes, removing the complete palindromic sequence plus adjacent nucleotides. At least the first type of deletion occurred in the absence of recA activity. Our results show a correlation between the sequence, as well as size, and the genetic stability of palindromes, i.e. sequences that could decrease the stability of a cruciform increased their genetic stability. This supports the theory that palindrome deletion occurs via extrusion of the palindrome into a cruciform or cruciform-like structure.     

Instability of a Plasmid-Borne Inverted Repeat in Saccharomyces Cerevisiae

Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes. We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S. cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division. Ten independent deletions had identical end points. Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat. The deletion rate and the types of deletions were unaffected by the rad52 mutation. Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat. The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.     

A family of repetitive palindromic sequences found in Neurospora mitochondrial DNA is also found in a mitochondrial plasmid DNA

Neurospora mtDNA contains a repetitive, 18 nucleotide palindromic sequence (5'-CCCTGCAGTACTGCAGGG-3') that contains two closely spaced PstI sites (CTGCAG) in the arms of the palindrome (Yin, S., Heckman, J., and RajBhandary, U. L. (1981) Cell 26, 325-332). In the present study, DNA sequence analysis was carried out to determine whether PstI palindromes are present in an apparently distinct genetic element, the 3.6-kilobase mitochondrial plasmid from Neurospora crassa strain Mauriceville-1c (FGSC 2225). The plasmid contains a cluster of closely spaced PstI sites extending over a 0.4-kilobase region (Collins, R. A., Stohl, L. L., Cole, M. D., and Lambowitz, A. M. (1981) Cell 24, 443-452). The DNA sequence shows that the cluster consists of eight PstI sites organized in five palindromic elements. Two of the elements are identical with the canonical sequence found in mtDNA, whereas the remaining three elements differ from the canonical sequence by a few nucleotides. The occurrence of the PstI palindromes in two otherwise unrelated DNA species is consistent with the hypothesis that they are related to mobile DNA sequences that either propagate or were once capable of propagating within mitochondria.     

A paucity of palindromes in φX174

The nucleotide sequence of the bacteriophage φX174 contains surprisingly few restriction endonuclease recognition sites. The observed frequency of those sites which consist of a six-nucleotide palindromic sequence would occur by chance with a probability of less than 8 × 10^?5. The genome of φX174 does not contain the four-nucleotide palindromic recognition site for the enzyme MboI and this finding has a probability of only 1·7 × 10^?9. A further analysis of the nucleotide sequence revealed that there is a marked scarcity of palindromic sequences with a length of four or six nucleotides while all other palindromes occur with a frequency close to that dictated by chance. A preliminary analysis of the genomes of other DNA viruses indicated that this palindrome avoidance is associated with single-stranded viruses only. The reasons for the paucity of these short even-numbered palindromes remains to be determined.     

The Effects of Nucleotide Sequence Changes on DNA Secondary Structure Formation in Escherichia Coli Are Consistent with Cruciform Extrusion in Vivo

The construction in bacteriophage λ of a set of long DNA palindromes with paired changes in the central sequence is described. Identical palindrome centers were previously used by others to test the S-type model for cruciform extrusion in vitro. Long DNA palindromes prevent the propagation of carrier phage λ on a wild-type host, and the sbcC mutation is sufficient to almost fully alleviate this inviability. The plaque areas produced by the palindrome containing phages were compared on an Escherichia coli sbcC lawn. Central sequence changes had a greater effect upon the plaque area than peripheral changes, implying that the residual palindrome-mediated inviability in E. coli sbcC is center-dependent and could be due to the formation of a cruciform structure. The results argue strongly that intrastrand pairing within palindromes is critical in determining their effects in vivo. In addition, the same data suggests that DNA loops in vivo may sometimes contain two bases only.     

Myc oncogene-induced genomic instability: DNA palindromes in bursal lymphomagenesis

Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic bic/miR-155 locus in all of our preneoplastic and neoplastic bursal samples, but not in DNA from normal and other transformed cell types. This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.     

Species-specific Typing of DNA Based on Palindrome Frequency Patterns

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics.     

Large DNA palindromes as a common form of structural chromosome aberrations in human cancers

Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.     

Palindrome resolution and recombination in the mammalian germ line.

Genetic instability is promoted by unusual sequence arrangements and DNA structures. Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination. We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level. A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems. In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements. Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination. Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated. Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome. Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion. By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable. Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells. Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome. We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.     

Phylogenetic analysis of heterocystous cyanobacteria (Subsections IV and V) using highly iterated palindromes as molecular markers

Highly iterated palindromes (HIP) have been used as high resolution molecular markers for assessing the genetic variability and phylogenetic relatedness of heterocystous cyanobacteria (subsections IV and V) representing 12 genera of heterocystous cyanobacteria, collected from different geographical areas of India. DNA fingerprints generated using four HIP markers viz. HIP-AT, HIP-CA, HIP-GC, and HIP-TG showed 100 % polymorphism in all the heterocystous cyanobacteria studied and each marker produced unique and strain-specific banding pattern. Furthermore, phylogenetic affinities based on the dendrogram constructed using HIP DNA profiles of heterocystous cyanobacteria suggest the monophyletic origin of this entire heterocystous clade along with a clear illustration of the polyphyletic origin of the branched Stigonematalean order (Subsection V). In addition, phylogenetic affinities were validated by principal component analysis of the HIP fingerprints. The overall data obtained by both the phylogeny and principal component assessments proved that the entire heterocystous clade was intermixed, and there are immediate needs for classificatory reforms that satisfy morphological plasticity and environmental concerns.     

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

Summary The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.     

Compositional Bias is a Major Determinant of the Distribution Pattern and Abundance of Palindromes in Drosophila melanogaster

Palindromic sequences are important DNA motifs related to gene regulation, DNA replication and recombination, and thus, investigating the evolutionary forces shaping the distribution pattern and abundance of palindromes in the genome is substantially important. In this article, we analyzed the abundance of palindromes in the genome, and then explored the possible effects of several genomic factors on the palindrome distribution and abundance in Drosophila melanogaster. Our results show that the palindrome abundance in D. melanogaster deviates from random expectation and the uneven distribution of palindromes across the genome is associated with local GC content, recombination rate, and coding exon density. Our data suggest that base composition is the major determinant of the distribution pattern and abundance of palindromes and the correlation between palindrome density and recombination is a side-product of the effect of compositional bias on the palindrome abundance.     

Role of neighbouring bases and assessment of strand specificity in ethylmethanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the SUP4-o gene of Saccharomyces cerevisiae

A total of 318 forward mutations induced by ethylmethanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the SUP4-o gene of the yeast Saccharomyces cerevisiae was characterized by DNA sequence analysis. Only base-pair substitutions were detected among the mutations examined and, for both agents, the majority (greater than 96%) were G.C to A.T. transitions. The remaining changes included A.T to G.C transitions and transversions at G.C sites. For EMS, two of the transversions were accompanied by nearby G.C to A.T transitions. There was considerable overlap of the sites within the SUP4-o gene that were mutated by EMS and MNNG and of the sites that each agent failed to mutate. However, EMS and MNNG mutagenesis differed with respect to the frequencies at which mutations were recovered at G.C pairs where the guanine is flanked (5') by a purine or pyrimidine. EMS exhibited no preference for either type of site, whereas a G.C site was 12-fold or fivefold more likely to be mutated by MNNG if preceded by a 5' adenine or guanine, respectively, than if flanked by a 5' pyrimidine. Finally, neither EMS nor MNNG mutagenesis showed a preference for G.C sites having the guanine on the non-transcribed strand.